anibiotics fok lecture 2008

7/27/2019 Anibiotics Fok Lecture 2008

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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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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)


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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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!)


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


Penicillin- the first antibiotic - 1928


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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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anibiotics fok lecture 2008

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