ANTIBIOTICS

Faculty of Dentistry 22 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

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)

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

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) - 1936 azo-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 Index
Chemotherapeutic Index = Toxic Concentration Effective Concentration

Chemotherapeutic index
DTM DCM DTM = dosis tolerata maxima (toxic) DCM = dosis curativa minima (effective) wide or narrow application concentration interval the larger, the better

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 use in a patient: 1942 World War II: saved 12-15% of lives

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

The Golden Age of Antibiotics

Discovery
1929 Penicillin Sulphonamides Gramicidin Penicillin Streptomycin & Bacitracin Cephalosporins Chloramphenicol & Chlorotetracycline Neomycin Trimethoprim Oxytetracycline Erythromycin Vancomycin Kanamycin Methicillin Ampicillin Cephalosporins Doxycycline Clindamycin Trimethoprim Tobramycin Cephamycins & Minocycline

Introduction

1930
1932 1939

1940
1942 1943 1945 1947 1949 1948

1950
1952 1956 1957

1960
1961 1963 1964 1966 1967 1968 Nalidixic acid Gentamicin

1970
1971 1972

PRINCIPLES OF ANTIBIOTIC TREATMENT

Principals of antibiotic treatment

Antibiotic

Bacterium

Wide or narrow spectrum Bacteriostatic or bactericid Penetration ability

Patient

Resistance !!!

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

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

Drugs in combination
10
7 6

Viable count per ml

Drug A + Drug B (Antagonism) Drug A Drug B

10 5 10 10 3 10 10 10
4

2 1

Drug A + Drug B (Synergy)

Aim of combinations
synergy
Sumetrolim: TMP + SMX Synercid: quinupristin + dalfopristin penicillin + gentamycin

avoiding resistance
-lactam + enzyme inhibitors

polymicrobial infection contraindicated:

-lactam + bacteriostatic !!
Acts only on multiplying bacteria Inhibits multiplication of bacteria

Pharmacological parameters

Pharmacokinetics: defines the time course

of drug absorption, distribution, metabolism and excretion.

Pharmacodynamics: refers to the

relationship between drug concentration at the site of action and pharmacologic response.

Pharmacokinetics
Dosing interval Dosing interval

Concentration (mg/L)

64 32 16 8 4 2 1

tmax

t Cmax

Time (hours)

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

Rabbit ears

Lysis

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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
lactam ring + 5 membered /=tiazolidin-/ ring with sulphur
N O

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

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Penicillin + enzyme inhibitor combination

enzyme inhibitor = lactam analogue ampicillin-sulbactam = Unasyn amoxicillin-clavulanic acid = Augmentin piperacillin-tazobactam = Tazocin
O S N O O N O O S N O

penicillin

clavulanic acid

sulbactam

I.1.2. Cephalosporins
lactam + 6 membered /=cephem-/ ring with sulphur

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

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I.1.3. Carbapenems
widest spectrum! derived from penicillins O imipenem, meropenem, ertapenem class B lactamase = carbapenemase
C N

I.1.4. Carbacephems
derived from cephalosporins loracarbef
N O

I.1.5. Monobactams
aztreonam
O

N SO3H

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 Gram-positives, locally by Bacillus subtilis bactericid, narrow spectrum

Polymixin:
desintegration of cell membrane Gram-negatives, locally (burns - Pseudomonas!) bactericid, narrow spectrum

II. Inhibition of protein synthesis (usually bacteriostatic)

tRNA

mRNA
30S

50S

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 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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II.3. Chloramphenicol
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

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
clindamycin, lincomycin

II.6. Streptogramins
quinupristin, dalfopristin in combination = Synercid

II.7. Ketolids
telithromycin

II.8. Oxazolidinons
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

Pteridine Para -aminobenzoic acid (PABA)
Dihydropteroic acid synthetase

Dihydropteroic acid Dihydrofolic acid

Dihydrofolic acid reductase (dhfr)

Sulphamethoxazole = PABA analogue bacteriostatic

Folic acid

Tetrahydrofolic acid DNA synthesis RNA synthesis Initiation of Protein synthesis Nucleotide synthesis Amino acid synthesis

Trimethoprim inhibits dhfr bactericid

In combination: Sumetrolim (1:5) co-trimoxazole (1:19)

III.3. Metronidazol
against anaerobes + some protozoa breaks down DNA
N 0 2N

CH3

CH2 CH2 OH

activated in the host cells by reduction of the nitro group at low redox potential (anaerobes!)

N - 0N

CH3

CH2 CH2 OH

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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 ineffective DNA
RNA DNA subunit (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% resistance to penicillin in Staphylococcus aureus 1935: discovery of sulphonamides 1950s: 50% resistance to sulphonamides in E. 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
vertical: spontaneous mutations (evolution, selection) normal mutation rate: 1 in 107 selection of resistant mutants:

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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chromosome R-plasmid

Plasmid transfer of antibiotic resistance genes

Bacterial cell resistant to ampicillin

sex pilus

Bacterial cell sensitive to ampicillin

Plasmid transfer of antibiotic resistance genes

Bacterial cell resistant to ampicillin

Bacterial cell RESISTANT to ampicillin

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Transposition - jumping genes

transposon

chromosome

plasmid

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 DRUG RESISTANCE !!!

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RESISTANCE MECHANISMS

The 3 major mechanisms

penicillins tetracyclines

sulphonamides

enzymatic inactivation altered target

active efflux

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1. Enzymatic inactivation - 1
cleaving (hydrolysis) of antibiotics !!
e.g. lactamase action on ampicillin:
HN
2

H N

H HN
2

HO

N O O N

S
OO O H

HO

N H COO-

COO-

-lactamase

-lactamases
very many different ~ mostly plasmid-encoded (sometimes chromosomal) constitutive or inducible (= in the presence of the lactam) ESBL:: extended spectrum lactamases !! TEM, SHV, CTX, OXA

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-lactamase classes
-lactamase Class

Class & Active Site Substrate

A Serine-

B Metallo-

C Serine-

D Serine-

Penicillins

Carbapenems

Cephalosporins

Penicillins

Chromosomal

Gram + & Gram -

Gram + & Gram -

Gram -

Gram -

Plasmid

Gram + & Gram -

Gram -

Gram -

Gram -

1. Enzymatic inactivation - 2
chemical modification: acetylation O2N adenylation phosphorylation Acetyl CoA methylation O2N aminoglycosides, chloramphenicol
Acetyl CoA
NH CO CH O Ac CCl 2 NH CH OH NH CH OH CH CH CO CH OH CO CH O Ac CCl 2 CCl 2

e.g. acetylation of chloramphenicol:

O2N

CH O Ac

CH

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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)

3. Efflux pump
removal of antibiotic not very effective 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
Chromosome

Plasmid DHFR TMP Dihydrofolate DHFR TMP

Tetrahydrofolate

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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 resistance

Impermeability Tetracycline Most antibiotics with Pseudomonas Tetracycline Fluoroquinolones -lactam ( -lactamases) Aminoglycosides (modifying enzymes) Trimethoprim Trimethoprim Sulphonamides Fluoroquinolones Aminoglycosides Efflux

Inactivation

Hyperproduction Altered Target

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Mechanisms of plasmid-mediated resistance

Inactivation -lactamases Aminoglycoside modifying enzymes
Acetyl transferases Adenyl transferases Phosphotransferases

Chloramphenicol acetyl transferase Efflux Tetracycline Chloramphenicol Trimethoprim Sulphonamides

Altered target

Problem bacteria
Staphylococcus aureus MRSA, VRSA (methicillin- and vancomycin resistance) Enterococcus faecalis and faecium VRE (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 streptomycinresistant 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 CLASS
rapidly 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) this is used in routine good for screening
inhibition zone

antibiotic often overestimates resistance discs

bacterium lawn

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 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: concentrationgradient on a strip
MIC

agar dilution (serial dilution of ab mixed into the medium) broth dilution (in tubes) or microdilution (96 well plates)

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MIC determination by dilution

0 1 2
Number of strains

R breakpoint
10 8 6 4 2 0 1 2 4 8 16 >16

8 (mg/L)

16

MIC (mg/L)

16

2 1 (mg/L)

0,5 0,25

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 strains: strains
ATCC: American Type Culture Collection NCTC: National Collection of Type Cultures

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Example: CLSI guidelines for Streptococcus pneumoniae

Incubation:
MH blood agar, at 352 oC, 5% CO2 suggested control strain: ATCC 49619

MIC breakpoints for penicillin:

S: 0,06 mg/L I: 0,125-1 mg/L R: 2 mg/L

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