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ANTIBIOTICSANTIBIOTICS
Faculty of Dentistry22 September 2008
Dobay Orsolya
Structure of the lecture
History of antibiotics
Principles of antibiotic treatment
Mode of actions of antibiotics
Resistance to antibiotics
Determination of antibiotic sensitivity
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HISTORY OF ANTIBIOTICS
History of antibiotics - 1
19th century:
Louis Pasteur & Robert Koch:
Bacteria as causative agents &Bacteria as causative agents &
recognisedrecognised need to control themneed to control them
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History of antibiotics - 2
First: plant extracts
Santonin (fromArtemisia maritima, the seawormwood)
againstAscaris, threadworm (helminths)
toxicity: disturbances of vision
Quinine (from bark of Cinchona tree)
against malaria (protozoon) cinchonism (impaired hearing, confusion)
Ipecacuanha root
(emetic used e.g. in dysentery)
History of antibiotics - 3
Toxic metals
Mercury
against syphilis (bacterial)
Arsenic
Atoxyl: for skin infections and Trypanosoma (1905)
TOO TOXIC ! (blindness)
Ehrlich: testing of several modified derivatives for
antimicrobial activity: no. 606 compound to test =
- Salvarsan (Arsphenamine) in 1910
- against Trypanosoma and syphilis
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History of antibiotics - 4
Dyes Paul Ehrlich:
noticed that parasites could be
distinguished from host tissue by dyes
Trypan Blue (dead cells will be blue) -
activity against Trypanosoma
Gerhard Domagk:
Prontosil (Sulphanilamide) - 1936azo-dye used in textile industry
1st synthetic antibacterial in general
clinical use (not an antibiotic!)
History of antibiotics - 5
Paul Ehrlich
started science of chemotherapy
selective toxicity !!
systematic chemical modifications(Magic Bullet)
developed the Chemotherapeutic IndexChemotherapeutic Index
Chemotherapeutic Index =Toxic Concentration
Effective Concentration
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Chemotherapeutic index
DTM = dosis tolerata maxima (toxic)
DCM = dosis curativa minima (effective)
wide or narrow application concentration
interval
DTM
DCMthe larger, the better
Alexander FlemingAlexander Fleming observed the
killing of staphylococci by a fungus
(Penicillium notatum)
observed by others - never exploited
Florey & Chain purified it by freeze-
drying (1940) -Nobel prize 1945
first use in a patient: 1942
World War II: saved 12-15% of lives
History of antibiotics - 6
Penicillin- the first antibiotic - 1928
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History of antibiotics - 7
Selman Waksman - Streptomycin (1943)
active against all Gram-negatives
first antibiotic active againstfirst antibiotic active against
Mycobacterium tuberculosisMycobacterium tuberculosis
most severe infections were caused by
Gram-negatives andMycobacterium
tuberculosis
extracted from Streptomyces
20 other antibiotics, incl. neomycin,
actinomycin
Nobel prize
1952
The Golden Age of AntibioticsDiscovery Introduction
1929 Penicillin
19301932 Sulphonamides
1939 Gramicidin
19401942 Penicillin
1943 Streptomycin&Bacitracin
1945 Cephalospor ins
1947 Chloramphenicol&Chlorotetracycline
1949 Neomycin
1948 Trimethoprim1950 Oxytetracycline
1952 Erythromycin
1956 Vancomycin
1957 Kanamycin
1960 Methicillin1961 Nalidixic acid Ampicillin
1963 Gentamicin
1964 Cephalosporins
1966 Doxycycline
1967 Clindamycin
1968 Trimethoprim
19701971 Tobramycin
1972 Cephamycins & Minocycline
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PRINCIPLES OF
ANTIBIOTIC TREATMENT
Principals of antibiotic treatment
Bacterium
Antibiotic
Patient
Resistance !!!
Wide or narrow
spectrum
Bacteriostatic or
bactericid
Penetration ability
Basic disease
Drug allergy
Pregnancy, childhood
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Types of antibiotic therapy
Targeted
based on sensitivity tests
Empiric
based on the symptoms and habits
knowledge of local epidemiological data
Profilactic
e.g. intestinal operation
Possible side effects
Allergy
penicillins!
type I hypersensitivity reaction (anaphylaxy)
Toxic effect kidney, liver (alcoholism!), bone marrow
hearing
bones, teeth
Disbacteriosis
= killing of the normal flora
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Drugs in combination
Drug A
Drug B
Drug A + Drug B(Antagonism)
Drug A + Drug B(Synergy)10
110
2
103
104
105
106
107
Viablecountperml
Aim of combinations synergy
Sumetrolim: TMP + SMX
Synercid: quinupristin + dalfopristin
penicillin + gentamycin
avoiding resistance
-lactam + enzyme inhibitors
polymicrobial infection
contraindicatedcontraindicated:
-lactam + bacteriostatic !!
Acts only on multiplying
bacteria
Inhibits multiplication of
bacteria
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Pharmacological parameters
Pharmacokinetics: defines the time courseof drug absorption, distribution, metabolism
and excretion.
Pharmacodynamics: refers to therelationship between drug concentration at
the site of action and pharmacologicresponse.
Pharmacokinetics
Concentration
(mg/L)
0 1 2 3 4 5 6
Time (hours)
tmax
t
Dosing interval Dosing interval
64
8
16
32
4
2
1
Cmax
Early antibiotics: short half-lives. Now: 1x a day is often enough!
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MODE OF ACTIONS OF
ANTIBIOTICS
Possible targets
Cell-wall synthesis
Cell membrane function
Protein synthesis
Folic acid synthesis
DNA synthesis
RNA synthesis
SELECTIVE TOXICITY !!!
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Cell
wall
Cell
membrane
I. Inhibition of cell wall synthesis
(bactericid)
Cell wall controls osmotic pressure
Filamentation
Lysis
Rabbit ears
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I.1. -lactams
Inhibit transpeptidation of
peptidoglycan chains
Important questions:
can be given orally? (acid stability)
lactamase (enzyme-) stability?
good against Gram negatives?
(Pseudomonas,Enterobacter!)
Structure of lactam ring:
(very sensitive!)
I.1.1. Penicillins
natural penicillins: penicillin G, V
enzyme stable: methicillin, oxacillin (MRSA!!)
amino-penicillins: ampicillin, amoxicillin
(givenper os, but not enzyme stable)
ureido-penicillins: piperacillin, mezlocillin (nor
acid or enzyme stable, but good against
Pseudomonas)
carboxi-penicillins: carbenicillin
lactam ring
+ 5 membered /=tiazolidin-/ ring with sulphurN
S
O
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Penicillin + enzyme inhibitor
combination
enzyme inhibitor = lactam analogue
ampicillin-sulbactam = Unasyn
amoxicillin-clavulanic acid = Augmentin
piperacillin-tazobactam = Tazocin
N
O
O
N
S
O
penicillin clavulanic acid
N
S
O
O O
sulbactam
I.1.2. Cephalosporins
more possibilities for substitution
also against Gram negatives!
class C lactamase = cephalosporinase
I. gen.: cefazolin, cephalexin, ...
II. gen: cefuroxim, cefaclor, cefoxitin, ...
III. gen.: cefotaxim, ceftriaxon,
IV. gen.: cefepim, cefpiron
lactam + 6 membered /=cephem-/ ring
with sulphur
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I.1.3. Carbapenems
widest spectrum!
derived from penicillins
imipenem, meropenem, ertapenem
class B lactamase = carbapenemase
I.1.4. Carbacephems
I.1.5. Monobactams
derived from cephalosporins
loracarbef
aztreonam
N
C
O
N
O SO3H
N
C
O
I.2. Glycopeptides
vancomycin, teicoplanin
giant molecules
triple effect:
cell wall synthesis membrane permeability
DNA synthesis (?)
last resort antibiotics
VRE!!VRE!!
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I.3. Polypeptides Bacitracin:
mainly Gram-positives, locally
byBacillus subtilis
bactericid, narrow spectrum
Polymixin:
desintegration ofcell membrane
Gram-negatives, locally (burns - Pseudomonas!)
bactericid, narrow spectrum
50S
II. Inhibition of protein synthesis
(usually bacteriostatic)
30S
tRNAmRNA
Aminoglycosides,
tetracyclines
Macrolides,
chloramphenicols
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II.1. Aminoglycosides
bactericid!
acts on 30S ribosomal subunit
streptomycin: also against TB (today: only)
today mainly:
amikacin, netilmycin: severe systemic infections
tobramycin, gentamycin: parenteral or eye drops
neomycin: eye drops
no full cross resistance
often toxicoften toxic (deafness!, kidney failure)
II.2. Tetracyclines chlortetracyclin, doxycyclin, oxytetracyclin
(Tetran)
act on 30S ribosomal subunit, inhibiting the
binding of aminoacil-tRNA
very wide spectrum (also for animals!) active against IC bacteria!!
Chlamydia,Mycoplasma,Rickettsia
side effects:
liver failure (pregnancy!), kidney failure
acculmulation in bones (teeth of children!)
severe diarrhoea, mucosal inflammation
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acts on 50S ribosomal subunit
Streptomyces venezuelae (Ehrlich)
wide spectrum dysbacteriosis !!
today mainly for:
typhus abdominalis, ampRHaem. influenzae
but: often in developing countries (cheap)
per os, or eye drops / ointments (Chlorocid) toxic effects:
bone marrow malfunction
Grey baby syndrome in newborns
II.3. Chloramphenicol
II.4. Macrolides
act on 50S ribosomal subunit
inhibit the elongation of peptide chain
higher concentration: becomes bactericid
groups:
14 membered ring: erythromycin, clarithromycin
15 membered ring : azythromycin
16 membered ring : josamycin
wide spectrum (Streptococci;Bordetella, STD, RTI/Haemophilus,pneumo/,Helicobacter, Chlamydia)
cross resistance exists!
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II.5. Lincosamides
quinupristin, dalfopristin
in combination = Synercid
II.6. Streptogramins
II.7. Ketolids
II.8. Oxazolidinons
clindamycin, lincomycin
telithromycin
linezolid
III. Inhibition of nucleic acid synthesis
III.1. Quinolons
inhibition of DNA gyrase
original compound: nalidixic acid
fluoroquinolones (FQs):
ciprofloxacin, ofloxacin, norfloxacin, sparloxacin
wide spectrum (also IC !)
newer FQs (wider spectrum, better activity)
mainly against Gram-positive upper RTI:
levofloxacin, moxifloxacin, gatifloxacin, gemifloxacin
Not in pregnancy or for young children!
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III.2. Inhibitors of folate synthesis
In combination:
Sumetrolim (1:5)
co-trimoxazole (1:19)
Para -aminobenzoic acid (PABA)Pteridine
DNA synthesis
RNA synthesis
Initiation of Protein synthesis
Nucleotide synthesis
Amino acid synthesis
Tetrahydrofolic acid
Dihydropteroic acid
Dihydrofolic acidFolic acid
Dihydropteroic acidsynthetase
Dihydrofolic acidreductase (dhfr)
Sulphamethoxazole
= PABA analogue
Sulphamethoxazole
= PABA analogue
bacteriostatic
TrimethoprimTrimethoprim
inhibits dhfrinhibits dhfr
bactericid
III.3. Metronidazol
against anaerobes + some protozoa
breaks down DNA
N CH3
CH CH OH2 2
0N-
N
N CH3
CH CH OH2 2
N
0 2N
activated in the host cells by
reduction of the nitro groupat low redox potential
(anaerobes!)
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III.4. RNA synthesis inhibition
Rifampin
inhibition of DNA dependent RNA
polymerase by binding to its subunit
if polymerisation has started already, it is
ineffectiveDNA
RNA
subunitDNA
(encoded by rpoB gene)
RESISTANCE TO
ANTIBIOTICS
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First emergence of resistance 1928: discovery of
penicillin
1940: first
identification of a -
lactamase
1945: 50% resistanceto penicillin in
Staphylococcus aureus
1935: discovery of
sulphonamides
1950s: 50% resistance
to sulphonamides inE.
coli
Natural resistance
against the antibiotic produced by themselves
cell wall barrier (Gram-negatives), or lack of
cell wall (Mycoplasma)
lack of transport system
lack of receptors
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Acquired resistance - 1 verticalvertical: spontaneous mutations (evolution,
selection)
normal mutation rate: 1 in 107
selection of resistant mutants:
Acquired resistance - 2
horizontalhorizontal: giving resistance genes to other
bacteria
by plasmid (conjugation)
by phage (transduction)
by transposon (mobile genetic elements)
by transformation (naked DNS)
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Bacterial
cell
sensitive toampicillin
Plasmid transferof antibiotic
resistance genes
Bacterial cell
resistant to
ampicillin
chromosome
R-plasmid
sex pilus
Bacterial cell
RESISTANT
to ampicillin
Bacterial cell
RESISTANT
to ampicillin
Plasmid transfer
of antibiotic
resistance genes
Bacterial cell
resistant to
ampicillin
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Transposition - jumping genes
chromosome plasmid
transposon
Transposons can acquire new genes by integrases
Human reasons leading to resistance
prescribing antibiotics too often
too long therapy, too low dose
stop taking the antibiotic before completing
the therapy
usage of antibiotics in animal husbandry
spread of resistant hospital strains (hygiene!)
MULTI DRUGMULTI DRUG
RESISTANCE !!!RESISTANCE !!!
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RESISTANCE MECHANISMS
The 3 major mechanisms
penicillins
enzymatic
inactivation
altered target
sulphonamides
tetracyclines
active
efflux
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1. Enzymatic inactivation - 1 cleaving (hydrolysis) of antibiotics !!
e.g. lactamaselactamase action on ampicillin:
HO
H NH
N
SN
O O
COO-
2
-lactamase
HO
HN H
N
S
N
OO
COO-
2
OH
H
-lactamases
very many different ~
mostly plasmid-encoded (sometimes
chromosomal)
constitutive or inducible (= in the presence ofthe lactam)
ESBL:ESBL:: extended spectrum lactamases !!
TEM, SHV, CTX, OXA
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-lactamase classes
-lactamaseClass
A
Serine-
Penicillins
Gram + & Gram -
B
Metallo-
Carbapenems
Gram + & Gram -
C
Serine-
Cephalosporins
Gram -
D
Serine-
Penicillins
Gram + & Gram - Gram - Gram - Gram -
Class &Active Site
Substrate
Chromosomal
Plasmid
Gram -
1. Enzymatic inactivation - 2
chemical modification:
acetylation
adenylation
phosphorylation
methylation
aminoglycosides,
chloramphenicol
H
N
O
2NO
2
CH CH CH
HO
H CClOC
H
N
O
2NO
2
CH CH CH
AcO
H CClOC
N
2NO
2
CH CH CH
H CClOC
Acetyl CoA
Acetyl CoA
e.g. acetylation of
chloramphenicol:
O OAcAc
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2. Alteration of target by mutation
decreased or no affinity
penicillins (pbp), aminoglycosides and
macrolides (30S and 50S ribosomal
subunits), quinolons (gyrA,B)
removal of antibiotic not very effective
macrolides, quinolons, tetracycline
3. Efflux pump
4. Overproduction of targets
e.g. overproduction of PABA (SMX)
5. Metabolite by-pass production of another target
e.g. an additional dihydrofolate reductase
DHFR
Plasmid
TMP
Dihydrofolate
Tetrahydrofolate
DHFR
Chromosome
TMP
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6. Change of membrane permeability blocking active transport
e.g. MRSA: altered membrane lipid
structure
e.g. tetracycline
7. Decreased modification to active
component
e.g. loss of nitrofurantoin-reductase
Mechanisms of chromosomal
resistanceImpermeability Tetracycline
Most antibiotics with Pseudomonas
Efflux TetracyclineFluoroquinolones
Inactivation -lactam (-lactamases)Aminoglycosides (modifying enzymes)
Hyperproduction Trimethoprim
Altered Target TrimethoprimSulphonamidesFluoroquinolones
Aminoglycosides
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Mechanisms of plasmid-mediatedresistance
Inactivation -lactamasesAminoglycoside modifying enzymes
Acetyl transferases
Adenyl transferases
Phosphotransferases
Chloramphenicol acetyl transferase
Efflux TetracyclineChloramphenicol
Altered target TrimethoprimSulphonamides
Problem bacteria
Staphylococcus aureusMRSA, VRSA
(methicillin- and vancomycin resistance)
Enterococcus faecalis andfaeciumVRE
(vancomycin resistance)
Carbapenem resistant Gram negatives
Acinetobacter baumannii
Pseudomonas aeruginosa
Klebsiella spp.
Stenotrophomonas maltophilia
MDR Mycobacterium tuberculosis
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Antibiotic resistant
Mycobacterium tuberculosis
21 January 1950
George Orwell died from an
untreatable streptomycin-
resistant strain of
Mycobacterium tuberculosis
Possible strategies to minimize
emergence of resistance
Avoiding the risks for cross-infection
Knowledge of the local sensitivity patterns
Always use the BEST IN CLASSrapidly bactericidal antibiotics are preferable
(dead bacteria cannot become resistant!)
avoid all use of slow-penetrating cephalosporins
Restricted use of carbapenems (mero-/imipenem)to otherwise untreatable bacteria
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DETERMINATION OF
ANTIBIOTIC SENSITIVITY
Disc diffusion test
Based on zone
diameter:
R(resistant)
I (intermediate)S (sensitive)
bacterium
lawnantibiotic
discs
inhibition zone
this is used in routine
often overestimates resistance
good for screening antibiogram
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Determination of MIC
definitions:
MIC = minimal inhibitory concentrationMIC = minimal inhibitory concentration
= the minimum concentration (in mg/L) of an
antibiotic enough to inhibit the growth of a
certain bacterial isolate
MBC = minimal bactericid concentration
at the population level:
MIC50, MC90: the conc. inhibiting 50/90% of all strains
MIC range: MIC interval between highest and lowest
values
Methods for MIC determination
Etest: concentration-
gradient on a strip
agar dilution (serial dilution of ab mixed into
the medium)
broth dilution (in tubes) or microdilution (96
well plates)
MIC
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1 2 4 8 16 >16
MIC (mg/L)
0
2
4
6
8
10
N
umber
ofstrains
R breakpoint
(mg/L)
0
4
1
8
2
16
MIC determination by dilution
2 1 0,5 0,254816
(mg/L)
Breakpoint determination
semi-quantitative
we check only at the resistance breakpoint
e.g.: screening for VRE: enterococci: vancomycin R = 8 mg/L
screening plate: with 6 mg/L vancomycin
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Sensitivity test guidelines
aim: standard results
national and international guidelines, e.g.
CLSI: Clinical and Laboratory Standard
Institute (American)
BSAC: British Society for Antimicrobial
Chemotherapy
EUCAST: European Committee on Antibiotic
Susceptibility Testing
Need for standardisation
breakpoint definitions (S, I, R)
continuous updates based on epedemiological
data
preparation of bacterial inoculum
incubation conditions (e.g. in air or CO2)
control strainscontrol strains:
ATCC: American Type Culture Collection
NCTC: National Collection of Type Cultures
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