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Antibiotic Resistance
Antibiotics were introduced as therapeutic agents against bacterial
disease starting in 1943
- Major classes of antibiotics attained widespread use by 1960’s
Infectious bacteria still a major health concern, especially in hospitals
- Post-operation infections by Staphylococcus aureus remain a
critical problem for surgery patients
In 1952, most Staph infections succumbed to penicillin
- By late 1960’s, Staph was resistant; next treated with methicillin
- By 1980’s, methicillin-resistance was widespread
- In 1990’s, vancomycin became “drug of last resort”
- Partial vancomycin resistance reported in late 90’s
- common in bacteria other than Staph…
Antibiotic Resistance
INTRODUCTION
1943
APPEARANCE
OF RESISTANCE
1946
Streptomycin
1945
1959
Tetracycline
1948
1953
Erythromycin
1952
1988
Vancomycin
1956
1988
Methicillin
1960
1961
Ampicillin
1961
1973
Cephalosporins
1964
late 1960’s
DRUG
Penicillin
Origins of Resistance
Drug resistance is a natural by-product of the evolutionary process:
natural selection acting on pre-existing genetic variation
400 microbial strains were isolated from natural sources and sealed
into vials in 1917, long before the clinical introduction of antibiotics
- Recent analysis: 11 of these 400 strains had antibiotic resistance
(at a low level)
-
Origins of Resistance
Nearly all clinically useful antibiotics are natural products, or their
synthetic derivatives; most were isolated from other microbes
- Fungi (penicillins, cephalosporins)
- Soil bacteria of genus Streptomyces (erythromycin, streptomycin,
tetracycline, vancomycin)
In 1999, only 1 class of antibiotic was totally synthetic (Ciprofloxacin)
Antibiotics are an ancient weapon...
Origins of Resistance
What does the antiquity of antibiotic resistance tell us?
- There is likely to be considerable genetic variation in natural
populations for genes that can potentially confer drug resistance
(i.e., the raw genetic material is there)
- Strong selection will quickly lead to the explosive growth of
resistant individuals, especially when most cells are susceptible
Widespread antibiotic use =
- nukes their competition
- the fittest survive and reproduce, passing on their resistance both
to clonal offspring and to other unrelated bacteria
Antibiotic Targets
The major classes of antibiotics affect 1 of 3 targets in bacteria cells:
(1) Cell wall biosynthesis
penicillins
(b-lactams)
cephalosporins
vancomycin
(non-ribosomal peptide)
(2) Protein synthesis
erythromycin (macrolide polyketides)
tetracycline (aromatic polyketides)
streptomycin, kanamycin (aminoglycosides)
(3) DNA replication
quinolones (Cipro)
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between
our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis
- block synthesis of peptidoglycan, the covalently cross-linked
peptide/glycan network, which imparts osmotic resistance to cell
(2) Protein synthesis
- target 23S rRNA + associated proteins in peptidyl transferase
center of bacterial ribosome
(3) DNA replication
- inhibit gyrase, essential enzyme that uncoils intertwined circles of
DNA after replication of the circular bacterial chromosome
Antibiotic Target 1: Cell Wall
Cell wall is peptidoglycan, a repeating polymer of di-saccharide,
tetra-peptide repeats cross-linked into a 3D matrix
b-lactam antibiotics interfere with cell wall biosynthesis of
Gram-positive bacteria (Staphylococci, Streptococci)
-
Antibiotic Target 1: Cell Wall
Bacterial transpeptidase enzyme forms crosslinking amide bonds
between #3 L-Lysine and #4 D-Alanine residues
TPase cuts off #5
D-Ala residue,
then links L-Lys
side chain to the
remaining D-Ala
Antibiotic Target 1: Cell Wall
Catalytic Serine -OH forms a temporary bond to the substrate
- when Lysine side-chain attacks the temp. ester linkage,
the Serine is restored to normal
-
Antibiotic Target 1: Cell Wall
b-lactams: Mechanism of Action
b-lactams inhibit transpeptidase by mimicking its substrate,
the terminal D-Ala—D-Ala
Transpeptidase attacks the b-lactam ring of penicillin, forms a
covalent bond that is slow to hydrolyze; enzyme is deactivated
Normally, the enzyme forms a temporary bond with D-Ala that
is rapidly broken by the side chain of Lysine
Resistance: b-lactamase Enzymes
Bacteria produce enzymes to hydrolyze the b-lactam ring before
drugs can reach inner membrane where PG synthesis occcurs
Resistance: b-lactamase Enzymes
A cell may produce 100,000 lactamase enzymes, each of which
can destroy 1,000 penicillins per second
100 million molecules of drug destroyed per second
Overcoming b-lactam Resistance
(resistance)
slow to
hydrolyze
(cell wall enz.)
Augmentin combines b-lactam antibiotic w/ clavulanate, a
“suicide” b-lactam that occupies the b-lactamase enzymes
- Allows active drug (amoxacillin) to reach target enzymes,
PG-synthesizing transpeptidases lining the inner membrane
Vancomycin: Mechanism of Action
Vancomycin, the crucial “drug of last resort,” inhibits PG synth
by binding directly to the D-Ala—D-Ala end of the peptide
- forms a cap over the end of the chain; blocks cross-linking
Vancomycin: Mechanism of Action
3D model of Vancomycin in
complex with D-Ala—D-Ala
note “cup-like”
shape of Van
Completely surrounds its target peptide, preventing enzymes
from reacting with the end of the peptidoglycan chain
Vancomycin
D-Ala
D-Ala
Vancomycin makes 5 H-bonds with the
D-Ala—D-Ala cap of the PG peptide
-
Van Resistance: D-Ala-D-Lactate
Vancomycin-resistant bacteria have peptidoglycan chains that end
in D-Ala—D-Lactate, instead of the usual D-Ala—D-Ala
(A) What genes are necessary to make this change?
(B) How does this confer resistance?
D-Ala—D-Ala
D-Ala—D-Lactate
Genetics of Van Resistance
5 gene products are required to produce Lac-terminal PG
- 2 “sensor” genes detect Van, turn on other 3 genes
- 2 synthesize the critical D-Ala—D-Lactate piece
- 1 destroys the pool of D-Ala—D-Ala in the cell (equilibrium)
VanH
VanA
reduction
VanX
hydrolysis
1,000 fold lower
affinity for Van
Vancomycin: Mechanism of Action
D-Ala—D-Ala
cap makes 5 H-bonds with Vancomycin
D-Ala—D-Lac
makes 1 less H-bond
Resistance
You die
Genetics of Van Resistance
Why did penicillin resistance appear in 2 years, but Van resistance
take 30 years to become a major health hazzard?
One answer: genetic complexity of resistance mechanism
Penicillin resistance requires the activity of one gene product
(b-lactamase enzyme)
- usually 2-4 year lag when only 1 gene is involved
Van resistance takes 5 gene products
- apparently delays development of infectious, highly resistant
strains when multiple gene products are involved
Overcoming Van Resistance
chlorinated
bi-phenyl
substituent
Approach #1: Screening of semi-synthetic analogues of Van
found that hydrophobic derivatives restore potentcy 100-fold
- Partitions drug to membrane surface, thus altering activity
and availability to target enzymes
Overcoming Van Resistance
Approach #2: Screening combinatorial libraries for novel small
molecules that cleave the D-Ala—D-Lac depsipeptide
- Look for drugs that can effectively function like an enzyme
Combinatorial library of 300,000 tripeptide derivatives yielded
3 hits, all w/ an N-terminal serine & an intramolecular H-bond
Pharmacophore deduced from computer modeling studies
HO
O
NH2
N
SProC5 “resensitized” bacteria
with Van-resistance, by cleaving
their D-Ala—D-Lac depsipeptide
SProC5
Chiosis & Boneca, Science 2001
Protein Synthesis Inhibitors
Tetracycline
(aromatic polyketide)
Erythromycin
(macrolide polyketide)
Kanamycin
(aminoglycoside)
Resistance to Aminoglycosides
(formerly a
protein kinase?)
Chemical modification of the drug lowers its
binding affinity for RNA target in the ribosome
-
MultiDrug Resistance Pumps
Bacteria use ATP-powered membrane proteins to pump any
lipophilic molecule out of the cell
- common in antibiotic-producing bacteria, to get drugs out
of their cells without poisoning themselves
Powerful method of resistance, because many different drugs
will be equally affected by these efflux pumps
MultiDrug Resistance Pumps
outside cell
(3) chamber then
opens, substrate
is expelled to
outer face of
membrane
(1) substrate binding:
lipophilic drug binds
inside cone-shaped
chamber; triggers
ATP hydrolysis
(2) chamber
then closes,
substrate flips
to opposite
orientation
Erythromycin Resistance
In addition to efflux pumps, erthyromycin resistance can arise
from reprogramming the target (akin to Van resistance)
Methylation of a specific adenine (#2058) on the 23S rRNA
component of the ribosome
- decreases binding affinity of erythromycin-class drugs
- does not impair protein synthesis
- present as a self-immunity mechanism in erythromycinproducing bacteria
Overcoming Erythromycin Resistance
Introduction of a 3-keto group into macrolide ring of
erythromycin class antibiotics alters conformation
- no induction of ribosome-methylating genes
- lower susceptibility to efflux by pumps
Erythromycin
Selection favoring Resistance
What causes the rapid occurrence of widespread resistance?
(1) Incomplete treatment: people fail to finish the full course of their
medication
- in the 1980’s, tuberculosis was almost wiped out w/ antibiotics
- in 1990’s, came back with a vengence, due to resistant strains
- 25% of previously-treated tuberculosis patients relapsed with drug
resistant strains; most had failed to complete their initial course
(2) Livestock doping: 50% of antibiotics used by livestock farmers
to increase yield of chicken, beef, pork
- high levels of antibiotics used in livestock result in strongly
resistant bacterial strains, which can then infect humans
Selection favoring Resistance
What causes the rapid occurrence of widespread resistance?
(3) Mis-prescription: my mom demands antibiotics for a cold
- widespread inappropriate use: up to 50% of prescriptions in
developing countries are for viral infections that cannot respond
(4) Gene transfer & multi-drug resistance
(a) genes encoding resistance accumulate on plasmids, transposons
confer simultaneous resistance to multiple drugs
(b) DNA is easily exchanged between unrelated bacteria
- vancomycin-resistant gut bacteria known since 1987
- resistance genes finally transferred to deadly infectious
Staphylococcus aureus in a Michigan hospital in 2002
Loss of Resistance...?
Resistance carries a cost: resistant bacteria grow more slowly under
normal conditions, pay a 10-20% fitness cost
- Replicating extra plasmid DNA is costly to the cell
- Ribosomal mutations that confer resistance slow protein production
When we stop using an antibiotic, does resistance go away?
- Can we reverse selection, and favor the vulnerable bacteria instead
Experiments show bacteria quickly evolve compensatory mutations
that lower the costs of resistance, instead of just losing resistance
-
Levin et al. 2000, Genetics 154: 985-997
Additional Reviews
Walsh, C.T. 2000. Molecular mechanisms that confer
antibacterial drug resistance. Nature 406: 775-781
Walsh, C.T. et al. 1996. Bacterial resistance to vancomycin:
five genes and one missing hydrogen bond tell the story.
Chemistry and Biology 3: 21-28
Davies, J. 1994. Inactivation of antibiotics and dissemination
of resistance genes. Science 264: 375-382.
Spratt, B.G. 1994. Resistance to antibiotics mediated by target
alterations. Science 264: 388-393.