introduction to antibiotics - kutatósemmelweis.hu/mikrobiologia/files/2014/09/fod_03.pdf• history...
TRANSCRIPT
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ANTIBIOTICS
Faculty of Dentistry
22 September 2014
Dobay Orsolya
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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
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History of antibiotics - 1
• 19th century:
– Louis Pasteur & Robert Koch:
Bacteria as causative agents &
recognized need to control them
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History of antibiotics - 2
• Plant extracts
– Quinine (against malaria)
– Ipecacuanha root (emetic, e.g. in dysentery)
• Toxic metals
– Mercury (against syphilis)
– Arsenic (Atoxyl, against Trypanosoma)
• Dyes
– Trypan Blue (Ehrlich)
– Prontosil (azo-dye, Domagk, 1936)
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History of antibiotics - 3
Paul Ehrlich
• started science of chemotherapy
• systematic chemical modifications
(“Magic Bullet”)
no. 606 compound = Salvarsan (1910)
• selective toxicity !!
• developed the Chemotherapeutic 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
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• Alexander 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 used in a patient: 1942
• World War II: penicillin saved 12-15% of lives
History of antibiotics - 4
Penicillin- the first antibiotic - 1928
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Fleming Museum, London
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History of antibiotics - 5
• Selman Waksman - Streptomycin (1943)
– active against all Gram-negatives
– first antibiotic active against
Mycobacterium tuberculosis
– most severe infections were caused by
Gram-negatives and Mycobacterium
tuberculosis
– extracted from Streptomyces
– 20 other antibiotics, incl. neomycin,
actinomycin
Nobel prize
1952
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PRINCIPLES OF
ANTIBIOTIC TREATMENT
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Principals of antibiotic treatment
Bacterium
Antibiotic
Patient
Gram + / -
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, dentical surgery
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Possible side effects• Allergy
– penicillins!
– type I hypersensitivity reaction (anaphylaxy)
• Toxic effect
– kidney, liver (alcoholism!), bone marrow
– impaired hearing
– bones, teeth (tetracyclin: complex with Ca2+)
– fluoroquinolones: Achilles-tendon rupture
• Disbacteriosis
= killing of the normal flora
– e.g. pseudomembranous colitis by C. difficile
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MODE OF ACTIONS OF
ANTIBIOTICS
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Possible targets• Inhibition of cell-wall synthesis
– inhibition of peptidoglycan cross-linking (beta-lactams)
– inhibition of peptidoglycan synthesis (vancomycin)
• Disruption of cell membrane– polymyxins
• Inhibition of protein synthesis– at 30S ribosomal subunit (aminoglycosides, tetracyclines)
– at 50S ribosomal subunit (macrolides, chloramphenicol)
• Inhibition of nucleic acid– inhibition of folic acid synthesis (sulphonamides,
trimethoprim)
– inhibition of DNA gyrase (fluoroquinolones)
– inhibition of RNA synthesis (rifampin)
SELECTIVE TOXICITY !!!
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Cell
wall
Cell
membrane
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I. Inhibition of cell wall synthesis
(bactericid)
Cell wall controls osmotic pressure
Filamentation
Lysis
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I.1. -lactams
• Inhibit transpeptidation of
peptidoglycan chains
• Important questions:
– can be given orally? (acid stability)
– β–lactamase (enzyme-) stability?
– good against Gram negatives?
(Pseudomonas, Acinetobacter!)
Structure of β–lactam ring:
(very vulnerable!)
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I.1.1. Penicillins
• natural penicillins: penicillin G, V
• enzyme stable: methicillin, oxacillin (MRSA!!)
• amino-penicillins: ampicillin, amoxicillin
(given per os, but not enzyme stable)
• ureido-penicillins: piperacillin, mezlocillin (nor
acid or enzyme stable, but good against
Pseudomonas)
• carboxi-penicillins: carbenicillin
β–lactam ring
+ 5 membered /=tiazolidin-/ ring with sulphur N
S
O
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I.1.2. Cephalosporins
• more possibilities for substitution
• also against Gram negatives!
• I. gen.: cefazolin, cephalexin, ...
• II. gen: cefuroxim, cefaclor, cefoxitin, ...
• III. gen.: cefotaxim, ceftriaxon, …
• IV. gen.: cefepim, cefpirom
• V. gen.: ceftaroline, ceftobiprol
β–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 β–lactamase = carbapenemase
I.1.4. Carbacephems
I.1.5. Monobactams
• derived from cephalosporins
• loracarbef
• aztreonam
N
C
O
N
O SO3H
N
C
O
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I.2. Glycopeptides
• vancomycin, teicoplanin
• giant molecules
• triple effect:
– cell wall synthesis
– membrane permeability
– DNA synthesis (?)
• last resort antibiotics
• VRE!!
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I.3. Polypeptides
• Bacitracin:
– mainly against S. aureus and Str. pyogenes, for
local treatment (skin infections)
– by Bacillus licheniformis
– inhibits cell wall synthesis
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II. Disruption of cell membrane
• Polymixins (e.g. Colistin):
– desintegration of cell membrane
– against Gram-negatives, for local treatment
– (burns, ear, eye - Pseudomonas!)
– bactericid, narrow spectrum
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50S
III. Inhibition of protein synthesis
(usually bacteriostatic)
α α
30S
tRNAmRNA
Aminoglycosides,
tetracyclines
Macrolides,
chloramphenicols
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III.1. Aminoglycosides
• bactericid!
• act on 30S ribosomal subunit
• streptomycin: also against TB (today: only)
• today mainly:
– amikacin, netilmycin: severe systemic infections
– tobramycin, gentamicin: parenteral or eye drops
– neomycin: eye drops
• often toxic (deafness!, kidney failure)
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III.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, ampR Haem. influenzae
• but: often in developing countries (cheap)
• per os, or eye drops / ointments (Chlorocid)
• toxic effects:
– bone marrow malfunction
– „Grey baby syndrome” in newborns
III.3. Chloramphenicol
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III.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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III.5. Lincosamides
• quinupristin, dalfopristin
• in combination = Synercid
III.6. Streptogramins
III.7. Ketolids
III.8. Oxazolidinons
• clindamycin, lincomycin
• telithromycin
• linezolid
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IV. Inhibition of nucleic acid synthesis
IV.1. Quinolons
• inhibition of DNA gyrase (supercoiling)
• original compound: nalidixic acid
• fluoroquinolones (FQs):
– ciprofloxacin, ofloxacin, norfloxacin, sparfloxacin
• 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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IV.2. Inhibitors of folate synthesis
In combination (1:5):
• Sumetrolim
• co-trimoxazole
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 acid synthetase
Dihydrofolic acid reductase (dhfr)
Sulphamethoxazole
= PABA analogue
bacteriostatic
Trimethoprim
inhibits dhfr
bactericid
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IV.3. Metronidazol
• against anaerobes + some protozoa
• directly 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 group
at low redox potential
(anaerobes!)
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IV.4. RNA synthesis inhibition
Rifampin
• inhibition of DNA dependent RNA polymerase
by binding to its β subunit
• if polymerisation has started already, it is
ineffective
• paints tear orange DNA
RNA
subunit DNA
(encoded by rpoB gene)
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Aim of combinations– synergy
• Sumetrolim: TMP + SMX
• Synercid: quinupristin + dalfopristin
• penicillin + gentamycin
– avoiding resistance
• ß-lactam + enzyme inhibitors
– polymicrobial infection
– contraindicated:
• ß-lactam + bacteriostatic !!
Acts only on multiplying
bacteria
Inhibits multiplication of
bacteria
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RESISTANCE TO
ANTIBIOTICS
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First emergence of resistance
• 1928: discovery of penicillin
• 1940: first identification of a β-lactamase
• 1945: 50% resistance to penicillin in
Staphylococcus aureus
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Antibiotic resistant
Mycobacterium tuberculosis
•1943: discovery of streptomycin
• 21 January 1950: George
Orwell died from an untreatable
streptomycin-resistant strain of
Mycobacterium tuberculosis
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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
• vertical: spontaneous mutations (evolution,
selection)
• normal mutation rate: 1 in 107
• selection of resistant mutants:
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Acquired resistance - 2
• horizontal: 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 to
ampicillin
Plasmid transfer
of antibiotic
resistance genes
Bacterial cell
resistant to
ampicillin
chromosome
R-plasmid
sex pilus
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Bacterial cell
RESISTANT
to ampicillin
Plasmid transfer
of antibiotic
resistance genes
Bacterial cell
resistant to
ampicillin
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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 DRUG
RESISTANCE !!!
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RESISTANCE MECHANISMS
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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. β–lactamase action on ampicillin:
HO
H NH
N
SN
OO
COO-
2
-lactamase
HO
H N H
N
S
N
OO
COO-
2
OH
H
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Penicillin + enzyme inhibitor
combination
• enzyme inhibitor = β–lactam analogue (suicidal molecules)
• 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
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β-lactamases
• very many different ~
• mostly plasmid-encoded (sometimes chromosomal)
• constitutive or inducible (= in the presence of the β–
lactam)
• ESBL: extended spectrum β–lactamases !!
TEM, SHV, CTX, OXA
by Gram negative bacteria
(E. coli, Klebsiella, Pseudomonas, Acinetobacter, …)
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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 (gyrase genes: gyrA,B)
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3. Efflux pump
• removal of antibiotic
• not very effective
• macrolides, quinolons, tetracycline
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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
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Problem bacteria
• Staphylococcus aureus – MRSA, VRSA
(methicillin- and vancomycin resistance)
• Enterococcus faecalis and faecium – VRE
(vancomycin resistance)
• MDR, XDR Mycobacterium tuberculosis
• Carbapenem resistant Gram negatives
– Acinetobacter baumannii
– Pseudomonas aeruginosa
– Klebsiella spp.
– Stenotrophomonas maltophilia
ESBL
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DETERMINATION OF
ANTIBIOTIC SENSITIVITY
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Disc diffusion test
• Based on zone
diameter:
– R (resistant)
– I (intermediate)
– S (sensitive)
bacterium
lawnantibiotic
discs
inhibition zone
• this is used in routine
• good for screening“antibiogram”
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Determination of MIC
• definitions:
– MIC = 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
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MIC determination by diffusion
• Etest: concentration-gradient on a strip
MIC
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MIC determination by dilution
2 1 0,5 0,254816
(mg/L)
(mg/L)
0
4
1
8
2
16agar dilution (AB mixed
into the medium)
broth dilution (AB mixed
into the medium)