antibiotics and antibiotic resistance dr. gerry wright · antibiotic discovery timeline β β 9...

Antibiotics and Antibiotic ResistanceDr. Gerry Wright

1The screen versions of these slides have full details of copyright and acknowledgements

Antibiotics and Antibiotic Resistance

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Gerry Wright Ph.DMcMaster University

Outline

• Antibiotics

– Classes

– Mode of action

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• Antibiotic Resistance

– Mechanisms

– Genetics

1 Antibiotics

3

1. Antibiotics



What are antibiotics?

• Small molecules that block the growth of microorganisms (bacteria and fungi)

• Growth inhibition can result in cell death (bactericidal)

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or simply stop cell growth (bacteriostatic)

Some definitions

Antibiotics: natural products that inhibit the growth of bacteria e.g. penicillin, erythromycin

Antibacterial agents: non-natural products that inhibit the growth of bacteria e.g. ciprofloxacin

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Anti-infective agents: compounds that inhibit microbial growth (antibiotics, antifungals, antiparasitic agents, antivirals)

Spectrum: Broad (many microbial species affected)Narrow (few microbial species affected)

How do we measure antibiotic activity?

• The MIC (Minimum Inhibitory Concentration) is a quantitative measure of antibiotic susceptibility

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• MICs can be measured on solid growth medium e.g. Kirby-Bauer disk assay, E-test, or in liquid broth microdilution assays E

- 32 --16-- 8 -- 4 -- 2 -- 1 --0.5-



Where do antibiotics come from?

• Antibiotics can be natural products (produced by other bacteria, fungi, plants, etc.) or be synthetic chemicals

• The first antibiotics were synthetic:

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y

– Salvarsan (1910) anti-syphilitic

– Sulfa-drugs (1935) antibacterial

• Paul Ehrlich’s concept of ‘magic bullet’ Paul Ehrlich

Antibiotics as natural products• The discovery of penicillin, a fungal metabolite

with antibacterial activity, in 1929 and it’s development as a drug in the early 1940s shifted the antibiotic focus to natural products

StreptomycinPenicillin

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p y

Fleming Florey Chain Waksman

Antibiotic discovery timeline

β

β

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β

“Golden Age” of antibiotic discovery

“Golden Age” of antibiotic medicinal chemistry



The consequence of antibiotic discovery

• ‘All the experts agree that by the year 2000, viral, and bacterial diseases will have been eradicated’Time, February, 1966

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• 1969, the US Surgeon General testified to congress that it was time to:

“Close the book on infectious diseases”

The surgeon general was wrong!‘New diseases’

Legionnaire’s (Legionella pneumophila)Lyme’s Disease (Borrelia burgdorferi)HIVSARS

‘Old diseases’

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Gastric ulcers & cancer (Helicobacter pylori)Tuberculosis (Mycobacterium tuberculosis)Influenza

BioterrorAnthrax (Bacillus anthracis)

Antibiotic resistanceMulti-drug resistant bacteria e.g. MRSA, VRE, VRSA

How do antibiotics work?

• To be clinically useful, antibiotics must inhibit the growth of microbes at concentrations that are not toxic to the host

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• Therefore, antibiotics usually target biology that is specific to microbes



Antibiotic targets: Gram positive

Cell wallMetabolism

Cell membrane

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Protein synthesis

DNA synthesis

RNA synthesis

Antibiotic targets: Gram negative

Cell wallMetabolism

Cell membrane

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Protein synthesis

DNA synthesis

RNA synthesis

Outer membrane

B. Protein Synthesis

Major antibiotic classes

A. Cell Wall

Tetracycline Aminoglycosides (gentamicin)

Macrolides(erythromycin)

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D. MetabolismC. DNA/RNA Synthesis

Fluroquinolones(ciprofloxacin) Rifampin

Trimethoprim Sulfamethoxazole

Beta-lactams e.g.penicillins, cephalosporins

Glycopeptides, e.g. vancomycin



2 Antibiotic resistance

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2. Antibiotic resistance

The problem

Antibiotic Discovery

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Antibiotic Resistance

Mechanisms of resistance

Permeability

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EffluxAlteredtargetEnzymes



Permeability

• Many antibiotics act on targets that are in the cytosol or the periplasm (Gram negatives), antibiotics therefore must cross a permeability barrier to enter the cell

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• Resistance as a result of permeability can be ‘passive’ e.g. the outer membrane of Gram negative bacteria

• Or active e.g. porin proteins, membrane potential, etc.

Antibiotic efflux• Once inside the cytosol or periplasmic space,

antibiotics can be actively ‘pumped’ from the cell using a variety of efflux proteins

• Ejection of antibiotics from inside the cell is coupled to ether H+ movement into the cell or ATP hydrolysis

• There are 5 major classes of antibiotic efflux systems

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• There are 5 major classes of antibiotic efflux systems in bacteria:

– Major Facilitator Subfamily (MFS)

– Small Multidrug Regulators (SMR)

– Resistance/Nodulation/Cell Division (RND)

– Multidrug and Toxin Extrusion (MATE)

– ATP-Binding Cassette (ABC)

MFS efflux• MFS efflux systems are widely distributed

in bacterial populations

• MFS associated genes are frequently found on mobile genetic elements (plasmids, transposons)

• They include the Quaternary Ammonium C d (Q ) t i T t ffl

Model of TetAA single polypeptide chain

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Compound (Qac) proteins, Tet efflux systems, NorA, and others

• These proteins consist of 12-14 transmembrane helices

• Models suggest that these have an inner core of 6 helices surrounding a water channel where antibiotics can penetrate and are exchanged for H+

Antibiotics penetrate the central water channel

H+



SMR efflux• These are small proteins (~12 kDa)

consisting of 4 transmembrane helices that likely operate as dimers

• Antibiotic efflux is coupled to H+ transport

Dimer modelof EmrE

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• The macrolide resistance protein EmrE is the prototype of the class for which there are cryo-EM and X-ray structural data supporting a model where 3 helices from each monomer form the pore and the 4th helix is required for dimer formation Antibiotics

H+

RND efflux• Tripartite efflux systems widely

distributed in Gram negative bacterial chromosomes

• AcrA-AcrB-TolC is the E. coliprototype and high resolution crystal structures are available

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crystal structures are available

• Inner membrane pore linked to an outer membrane pore via a periplasmic linker protein

• Antibiotic efflux coupled to H+ influx

• MexA-B,OprM and several other in Pseudomonas aeruginosa Antibiotics

H+

ABC efflux

• Common bacterial efflux system

• ABC efflux systems couple ATP hydrolysis with antibiotic export

• These efflux systems are heterodimeric complexes of a transmembrane

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complexes of a transmembrane membrane pore with a cytoplasmic ATP-hydrolyzing subunit

• ABC systems are common components of antibiotic biosynthetic gene clusters in producing bacteria

• Commonly chromosomally encoded

Antibiotics

ATP

ADP



Antibiotic modifying enzymes

• Modification or destruction of antibiotics is a common mechanism of resistance

• Mechanisms include:

– Hydrolysis e.g. β-lactams

– Reduction/oxidation e g tetracyclines

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– Reduction/oxidation e.g. tetracyclines

– Chemical modifications such as:• Acetylation

• Phosphorylation

• Nucleotidylation

• ADP-ribosylation

• …

β-Lactams

N

S

OCOOH

HHNR

O

H

Penicillin

NO

HHNR1

O

HS

COOHCephalosporin

R2

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NO

COOH

HR1

H

R2

Carbapenen

N SO3HO

HHN

R2

Monobactam

R1

O

N

O

OCOOH

R

Clavam

β-Lactamases

Ambler Notation

A. Penicillinases (TEM, SHV, KPC, CTX-M)

B Metallo (IMP VIM)

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B. Metallo (IMP, VIM)

C. Cephalosporinases (AmpC)

D. Oxacillinases (OXA)



Mechanisms

Class A,C,D

H2O

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Class B Inactive antibiotic

HO - Zn

ESBLs

• Class A enzymes

• 292 reported (www.lahey.org/Studies/)

• Characterized by resistance

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to extended spectrum β-lactams (cefotaxime, ceftriaxone, ceftazidime, aztreonam, etc.)

• Mutations in TEM, SHV and CTX-M enzymes

KPC β-Lactamases

• Class A Carbapenemase producing Klebsiella

• Can transfer to E. coli, Enterobacter, Salmonella

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• Emerged in 1990s, but recent outbreaks worldwide (New York, Israel)

• 5 isozymes now known

• Pan-resistant strains



Metallo β-Lactamases

• IMP,SPM,GIM & VIM predominate

C f i t

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• Confer resistance to all β-lactams

• No inhibitors

Aminoglycosides

ANT

ATP PPI

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• Enzymes

– APH (Kinase)

– ANT (adenyltransferase)

– AAC (acetyltransferase)

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APH AAC ANT



Tetracyclines

• TetR-TetA (regulated efflux)

• TetM (ribosomal protection)

• TetX (enzymatic inactivation)

34Biochemistry (2005) 44:11829

OH O O

OHH H

N(CH3)2OH

NH2

O

N(CH3)2

OHNH

HN

O

OH O O

OHH H

N(CH3)2OH

NH2

O

N(CH3)2

ONH

HN

O

OH

TetX

NADPHO2

Rifamycins• Mutation in RpoB

• Arr ADP-ribosyltransfase

35PNAS (2008) 105:4886

Antibiotic resistance by target modification

• Masking the molecular target of antibiotics is an effective mechanism of resistance

• Examples include Erm-mediate methylation

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of bacterial ribosome 23S rRNA that blocks the binding site for macrolide, lincosamide and type B streptogramins

• High level resistance to glycopeptide antibiotics such as vancomycin is another example



Glycopeptide antibiotics

O

OCl

O

HO Cl OH

OHO

HOHO

OMeOH

NH3+

MeO

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N N NO

ONH2

+NNH

O HN

O

O

O

HO OH

-OO

Me

O NH2

OH

ON

NH

R

O-

O

O

HH H

H

H

OH

Glycopeptide resistance in VRE

OOHOHO

OMeOH

NH3+

MeO

vanR vanS vanH vanA vanX

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O

O

N N

Cl

NO

ONH2

+

O

NNH

HOO H

N

O

O

O

HO OH

-O

Cl

O

Me

O NH2

OH

HOHO

HH H H

OH

OO

NH

R

O-

O

O

ON

NH

R

O-

O

O H

Antibiotic resistance by other means

• Selection of mutants e.g. mutations in GyrA/ParC for fluroquinolone antibiotics

• Expression of immunity proteins e.g. QnR

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for fluroquinolone antibiotics

• Metabolic bypass by insensitive variantse.g. trimethoprim, sulfa-drugs



Quinolones

• Mutations in GyrA, ParC

• Plasmid mediated Qnr

• AAC(6’)-Ib-cr

NN

F

HN

OO

OH

NN

F

HN

OO

OH

Norfloxacin Ciprofloxacin

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• AAC(6 )-Ib-cr

NN

F

HN

OO

OH

H3CONN

F

N

OO

OH

O

Gatifloxacin Levofloxacin

Trimethoprim-Sulfa

• Biosynthetic bypass

• Mutations in target metabolic enzymes DHFR, dihydropteroate synthase (Sul)

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• Plasmid mediated

MDR = combinatorial resistance

Permeability Enzymes

Drug

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Target Efflux

Biofilm



“…pitted against microbial genes we have mainly our wits”

Joshua LederbergJAMA (1996) 276:418

There is a great need for new antimicrobials

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JAMA (1996) 276:418

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