ANTIBIOTICS

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ANTIBIOTICS

ANTIBIOTICS

Faculty of Dentistry

24 September 2012

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


HISTORY OF ANTIBIOTICS


History of antibiotics - 1

• 19th century:

– Louis Pasteur & Robert Koch:

Bacteria as causative agents &

recognised need to control them


History of antibiotics - 2

First: plant extracts

• Santonin (from Artemisia maritima, the “sea

wormwood”)

→ against Ascaris, 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


• Mercury

History of antibiotics - 3

Toxic metals

→ against syphilis (bacterial)

• Arsenic

→ Atoxyl: for skin infections and Trypanosoma (1905)

TOO TOXIC ! (blindness)

Ehrlich: testing of several modified derivatives for

antimicrobial activity:

- Salvarsan (Arsphenamine) in 1910

- against Trypanosoma and syphilis


• Paul Ehrlich:

History of antibiotics - 4

Dyes

– 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) - 1936

– azo-dye used in textile industry

– 1 st synthetic antibacterial in general

clinical use (not an antibiotic!)


History of antibiotics - 5

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


Chemotherapeutic index

DTM

DCM

the larger, the better

• DTM = dosis tolerata maxima (toxic)

• DCM = dosis curativa minima (effective)

• wide or narrow application concentration

interval


History of antibiotics - 6

Penicillin- the first antibiotic - 1928

• Alexander Fleming observed the

killing of staphylococci by a fungus

(Penicillium notatum)

• observed by others - never exploited

• Florey & Chain purified it by freezedrying

(1940) - Nobel prize 1945

• first used in a patient: 1942

• World War II: penicillin saved 12-15%

of lives


Fleming Museum, London


History of antibiotics - 7

• 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


PRINCIPLES OF

ANTIBIOTIC TREATMENT


Principals of antibiotic treatment

Bacterium

Gram + / -

Resistance !!!

Antibiotic

•Wide or narrow

spectrum

•Bacteriostatic or

bactericid

•Penetration ability

Patient

•Basic disease

•Drug allergy

•Pregnancy, childhood


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


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

– e.g. pseudomembranous colitis by C. difficile


MODE OF ACTIONS OF

ANTIBIOTICS


Possible targets

• Inhibition of cell-wall 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 nucleic

acid

– inhibition of folic acid synthesis (sulphonamides,

trimethoprim)

– inhibition of DNA gyrase (fluoroquinolones)

– inhibition of RNA synthesis (rifampin)

SELECTIVE TOXICITY !!!


Cell

wall

Cell

membrane


I. Inhibition of cell wall synthesis

Cell wall controls osmotic pressure

(bactericid)

Filamentation

“Rabbit ears”

Lysis


I.1. β-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!)


I.1.1. Penicillins

β–lactam ring

+ 5 membered /=tiazolidin-/ ring with sulphur

• 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

O

N

S


I.1.2. Cephalosporins

β–lactam + 6 membered /=cephem-/ ring

with sulphur

• more possibilities for substitution

• also against Gram negatives!

• I. gen.: cefazolin, cephalexin, ...

• II. gen: cefuroxim, cefaclor, cefoxitin, ...

• III. gen.: cefotaxim, ceftriaxon, …

• IV. gen.: cefepim, cefpiron


I.1.3. Carbapenems

• widest spectrum!

• derived from penicillins

• imipenem, meropenem, ertapenem

• class B β–lactamase = carbapenemase

I.1.4. Carbacephems

• derived from cephalosporins

• loracarbef

• aztreonam

I.1.5. Monobactams

O

O

N

N

N

C

C

O SO 3 H


I.2. Glycopeptides

• vancomycin, teicoplanin

• giant molecules

• triple effect:

– cell wall synthesis

– membrane permeability

– DNA synthesis (?)

• last resort antibiotics

• VRE!!


• Bacitracin:

I.3. Polypeptides

– mainly against S. aureus and Str. pyogenes, for

local treatment (skin infections)

– by Bacillus licheniformis

– inhibits cell wall synthesis


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


III. Inhibition of protein synthesis

tRNA

(usually bacteriostatic)

α α

Aminoglycosides,

tetracyclines

30S

50S

mRNA

Macrolides,

chloramphenicols


• bactericid!

III.1. Aminoglycosides

• acts 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)


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


III.3. Chloramphenicol

• acts on 50S ribosomal subunit

• Streptomyces venezuelae (Ehrlich)

• wide spectrum → dysbacteriosis !!

• today mainly for:

– typhus abdominalis, amp R 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.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!


III.5. Lincosamides

• clindamycin, lincomycin

III.6. Streptogramins

• quinupristin, dalfopristin

• in combination = Synercid

• telithromycin

• linezolid

III.7. Ketolids

III.8. Oxazolidinons


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!


IV.2. Inhibitors of folate synthesis

Folic acid

Pteridine

Para -aminobenzoic acid (PABA)

Dihydropteroic acid

synthetase

Dihydropteroic acid

Dihydrofolic acid

Dihydrofolic acid

reductase (dhfr)

Tetrahydrofolic acid

DNA synthesis

RNA synthesis

Initiation of Protein synthesis

Nucleotide synthesis

Amino acid synthesis

Sulphamethoxazole

= PABA analogue

bacteriostatic

Trimethoprim

inhibits dhfr

bactericid

In combination (1:5):

• Sumetrolim

• co-trimoxazole


IV.3. Metronidazol

• against anaerobes + some protozoa

• breaks down DNA

0 2N

- 0N

N

N

N

N

CH 3

CH CH OH

2 2

CH 3

CH CH OH

2 2

• activated in the host cells by

reduction of the nitro group

at low redox potential

(anaerobes!)


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

β subunit

(encoded by rpoB gene)

DNA

RNA

DNA


Viable count per ml

10 7

10 6

10 5

10 4

10 3

10 2

10 1

Drugs in combination

Drug A + Drug B

(Antagonism)

Drug A

Drug B

Drug A + Drug B

(Synergy)


– synergy

Aim of combinations

• Sumetrolim: TMP + SMX

• Synercid: quinupristin + dalfopristin

• penicillin + gentamycin

– avoiding resistance

• ß-lactam + enzyme inhibitors

– polymicrobial infection

– contraindicated:

contraindicated

• ß-lactam + bacteriostatic !!

Acts only on multiplying

bacteria

Inhibits multiplication of

bacteria


RESISTANCE TO

ANTIBIOTICS


First emergence of resistance

• 1928: discovery of penicillin

• 1940: first identification of a β-lactamase

• 1945: 50% resistance to penicillin in

Staphylococcus aureus


Antibiotic resistant

Mycobacterium tuberculosis

21 January 1950

George Orwell died from an

untreatable streptomycinresistant

strain of

Mycobacterium tuberculosis


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


Acquired resistance - 1

• vertical: vertical spontaneous mutations (evolution,

selection)

• normal mutation rate: 1 in 10 7

• selection of resistant mutants:


Acquired resistance - 2

• horizontal: horizontal giving resistance genes to other

bacteria

– by plasmid (conjugation)

– by phage (transduction)

– by transposon (mobile genetic elements)

– by transformation (naked DNS)


Bacterial cell

resistant to

ampicillin

chromosome

R-plasmid

sex pilus

Plasmid transfer

of antibiotic

resistance genes

Bacterial

cell

sensitive to

ampicillin


Bacterial cell

resistant to

ampicillin

Plasmid transfer

of antibiotic

resistance genes

Bacterial cell

RESISTANT

to ampicillin


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 !!!


RESISTANCE MECHANISMS


sulphonamides

The 3 major mechanisms

penicillins

altered target

enzymatic

inactivation

tetracyclines

active

efflux


HO

1. Enzymatic inactivation - 1

• cleaving (hydrolysis) of antibiotics !!

– e.g. β–lactamase lactamase action on ampicillin:

H N

2

O

H

N

O

N

S

COO -

β-lactamase

HO

H N

2

O O

O H

N

H

N

H

S

COO -


Penicillin + enzyme inhibitor

combination

• enzyme inhibitor = β–lactam analogue

(suicidal molecules)

• ampicillin-sulbactam = Unasyn

• amoxicillin-clavulanic acid = Augmentin

• piperacillin-tazobactam = Tazocin

O

N

S

penicillin clavulanic acid

O

N

O

O

O O

N

S

sulbactam


• very many different ~

β-lactamases

• 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, …)


Class &

Active Site

Substrate

Chromosomal

Plasmid

β-lactamase classes

A

Serine-

Penicillins

Gram + & Gram -

B

Metallo-

Carbapenems

Gram + & Gram -

β-lactamase

Class

C

Serine-

Cephalosporins

Gram -

D

Serine-

Penicillins

Gram -

Gram + & Gram - Gram - Gram - Gram -


1. Enzymatic inactivation - 2

• chemical modification:

– acetylation

– adenylation

– phosphorylation

– methylation

• aminoglycosides,

chloramphenicol

e.g. acetylation of

chloramphenicol:

2N O

Acetyl CoA

2N O

Acetyl CoA

2N O

OH

NH

CO

CCl

CH CH CH

OH

OH

NH

CO

CCl

CH CH CH

O

Ac

NH

CO

CCl

CH CH CH

OAc OAc

2

2

2


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)


• removal of antibiotic

• not very effective

3. Efflux pump

• macrolides, quinolons, tetracycline


4. Overproduction of targets

• e.g. overproduction of PABA (SMX)

5. Metabolite by-pass

• production of another target

– e.g. an additional dihydrofolate reductase

TMP

DHFR

Chromosome

Dihydrofolate

Tetrahydrofolate

DHFR

Plasmid

TMP


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


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


DETERMINATION OF

ANTIBIOTIC SENSITIVITY


• Based on zone

diameter:

– R (resistant)

– I (intermediate)

– S (sensitive)

• this is used in routine

• good for screening

Disc diffusion test

antibiotic

discs

“antibiogram”

inhibition zone

bacterium

lawn


• definitions:

Determination of MIC

– 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

• at the population level:

– MIC 50, MC 90: the conc. inhibiting 50/90% of all strains

– MIC range: MIC interval between highest and lowest

values


MIC determination by diffusion

• Etest: concentration-gradient on a strip

MIC


16

0

4

MIC determination by dilution

8

8

(mg/L)

4

1

2 1 0,5 0,25

(mg/L)

2

16

agar dilution (AB mixed

into the medium)

broth dilution (AB mixed

into the medium)


Additional material


1930

1940

1950

1960

1970

The Golden Age of Antibiotics

Discovery Introduction

1929 Penicillin

1932 Sulphonamides

1939 Gramicidin

1942 Penicillin

1943 Streptomycin & Bacitracin

1945 Cephalosporins

1947 Chloramphenicol & Chlorotetracycline

1949 Neomycin

1948 Trimethoprim

Oxytetracycline

1952 Erythromycin

1956 Vancomycin

1957 Kanamycin

Methicillin

1961 Nalidixic acid Ampicillin

1963 Gentamicin

1964 Cephalosporins

1966 Doxycycline

1967 Clindamycin

1968 Trimethoprim

1971 Tobramycin

1972 Cephamycins & Minocycline


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 CO 2 )

• control strains: strains

– ATCC: American Type Culture Collection

– NCTC: National Collection of Type Cultures


Example: CLSI guidelines for

Streptococcus pneumoniae

• Incubation:

– MH blood agar, at 35±2 o C, 5% CO 2

– suggested control strain: ATCC 49619

• MIC breakpoints for penicillin:

– S: ≤ 0,06 mg/L

– I: 0,125-1 mg/L

– R: ≥ 2 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


Number of strains

10

8

6

4

2

0

R breakpoint

1 2 4 8 16 >16

MIC (mg/L)

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