antibiotic resistance. antibiotic resistance antibiotics were introduced as therapeutic agents...
TRANSCRIPT
Antibiotic resistance
Antibiotic ResistanceAntibiotics 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 APPEARANCE
DRUG INTRODUCTION OF RESISTANCE
Penicillin 1943 1946
Streptomycin 1945 1959
Tetracycline 1948 1953
Erythromycin 1952 1988
Vancomycin 1956 1988
Methicillin 1960 1961
Ampicillin 1961 1973
Cephalosporins 1964 late 1960’s
Origins of ResistanceDrug 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)
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Origins of ResistanceNearly 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 ResistanceWhat 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 =
-competition - the fittest survive and reproduce, passing on their resistance both to clonal offspring and to other unrelated bacteria
Antibiotic TargetsThe major classes of antibiotics affect 1 of 3 targets in bacteria cells:
(1) Cell wall biosynthesis
penicillins cephalosporinsvancomycin (non-ribosomal peptide)
(2) Protein synthesis
erythromycin (macrolide polyketides)tetracycline (aromatic polyketides)streptomycin, kanamycin (aminoglycosides)
(3) DNA replication
quinolones (Cipro)
(-lactams)
Antibiotic TargetsAntibiotics 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 WallCell wall is peptidoglycan, a repeating polymer of di-saccharide, tetra-peptide repeats cross-linked into a 3D matrix
-lactam antibiotics interfere with cell wall biosynthesis of Gram-positive bacteria (Staphylococci, Streptococci)
-
Antibiotic Target 1: Cell WallBacterial 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
-lactams: Mechanism of Action
-lactams inhibit transpeptidase by mimicking its substrate, the terminal D-Ala—D-Ala
Transpeptidase attacks the -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: -lactamase Enzymes
Bacteria produce enzymes to hydrolyze the -lactam ring before drugs can reach inner membrane where PG synthesis occcurs
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
Resistance: -lactamase Enzymes
Overcoming -lactam Resistance
Augmentin combines -lactam antibiotic w/ clavulanate, a “suicide” -lactam that occupies the -lactamase enzymes
- Allows active drug (amoxacillin) to reach target enzymes, PG-synthesizing transpeptidases lining the inner membrane
(resistance) slow tohydrolyze
(cell wall enz.)
Vancomycin: Mechanism of ActionVancomycin, 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
Completely surrounds its target peptide, preventing enzymes from reacting with the end of the peptidoglycan chain
3D model of Vancomycin incomplex with D-Ala—D-Ala
note “cup-like” shape of Van
Vancomycin makes 5 H-bonds with the D-Ala—D-Ala cap of the PG peptide
- -
Vancomycin
D-Ala D-Ala
Van Resistance: D-Ala-D-LactateVancomycin-resistant bacteria have peptidoglycan chains that end in D-Ala—D-Lactate, instead of the usual D-Ala—D-Ala
D-Ala—D-Ala
D-Ala—D-Lactate
Genetics of Van Resistance
VanAVanH
VanX
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)
reduction
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 death
Genetics of Van ResistanceWhy 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 (-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
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
chlorinated bi-phenyl substituent
Overcoming Van ResistanceApproach #2: Screening combinatorial libraries for novel small molecules that cleave the D-Ala—D-Lac peptide
- 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
N
HO
NH2
OSProC5 “resensitized” bacteria with Van-resistance, by cleaving their D-Ala—D-Lac
SProC5
Chiosis & Boneca, Science 2001
Protein Synthesis Inhibitors
Erythromycin (macrolide polyketide)
Tetracycline (aromatic polyketide)
Kanamycin(aminoglycoside)
Resistance to Aminoglycosides
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
(1) substrate binding: lipophilic drug binds inside cone-shaped chamber; triggers ATP hydrolysis
(2) chamber then closes, substrate flips to opposite orientation
(3) chamber then opens, substrate is expelled to outer face of membrane
outside cell
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 erythromycin- producing 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 ResistanceWhat 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 ResistanceWhat 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
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Levin et al. 2000, Genetics 154: 985-997
Additional ReviewsWalsh, 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.