Antibiotics and Antibiotic ResistanceDr. Gerry Wright
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Antibiotics and Antibiotic Resistance
1
Gerry Wright Ph.DMcMaster University
Outline
• Antibiotics
– Classes
– Mode of action
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• Antibiotic Resistance
– Mechanisms
– Genetics
1 Antibiotics
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1. Antibiotics
Antibiotics and Antibiotic ResistanceDr. Gerry Wright
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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-
Antibiotics and Antibiotic ResistanceDr. Gerry Wright
3The screen versions of these slides have full details of copyright and acknowledgements
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
9
β
“Golden Age” of antibiotic discovery
“Golden Age” of antibiotic medicinal chemistry
Antibiotics and Antibiotic ResistanceDr. Gerry Wright
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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
Antibiotics and Antibiotic ResistanceDr. Gerry Wright
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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
Antibiotics and Antibiotic ResistanceDr. Gerry Wright
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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
Antibiotics and Antibiotic ResistanceDr. Gerry Wright
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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+
Antibiotics and Antibiotic ResistanceDr. Gerry Wright
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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
Antibiotics and Antibiotic ResistanceDr. Gerry Wright
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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)
Antibiotics and Antibiotic ResistanceDr. Gerry Wright
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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
Antibiotics and Antibiotic ResistanceDr. Gerry Wright
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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
Antibiotics and Antibiotic ResistanceDr. Gerry Wright
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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
Antibiotics and Antibiotic ResistanceDr. Gerry Wright
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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
Antibiotics and Antibiotic ResistanceDr. Gerry Wright
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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
Antibiotics and Antibiotic ResistanceDr. Gerry Wright
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“…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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