Dynamics of Antibiotic Resistance Evolution
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Transcript of Dynamics of Antibiotic Resistance Evolution
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Dynamics of Resistance Evolution
Lourens Robberts, PhD. 2007
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How does resistance become a problem for patients?
Ü Acquisition of already resistant pathogen from environment
Ü Selection of already resistant strains from within patient (enrichment)
Ü Imposing antimicrobial pressure on wild-type (sensitive) pathogen to create the necessary conditions for evolution of resistance mutations
Ü Imposing pressure on wild-type pathogen to acquire and maintain exogenous resistance genes
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Bacterial genetics:
Conjugative transfer Transformation,
Mutation
NEW BACTERIAL
RESISTANCE
Human investment:
R & D
NEW ANTIBIOTIC
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Ü Phenotype (observable resistance mechanisms) derive from the genotype
Ü How does a new genotype (new genes/altered genes) arise that enables defence against antimicrobials?
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Survival of GENES
Epidemic spread of clones viz GENES
Development of a mutation in the gene
Spread of the gene among hosts (bacteria), and spread of hosts with new gene
Environmental pressure on the host
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Mutations of pre-existing genetic determinants
Type of mutation Resistance phenotype
Structural Streptomycin Rifampicin Fluoroquinolones Sulfonamides Trimethoprim
Regulatory Aminoglycosides (aarA) β-lactamases (AmpC) Chloramphenicol Fluoroquinolones Tetracyclines (marA) Imipenem (OMP)
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Acquisition of foreign DNA Phenotype Acquired genes
Aminoglycosides Aminoglycoside-modifying enzymes
β-lactams β-lactamase genes
Chloramphenicol CAT genes
Erythromycin/clindamycin Methylase/MLSB genes
Methicillin mecA gene
Penicillin PBP genes, β-lactamase
Sulfa DHPS gene
Trimethoprim DHFR gene
Tetracyclines Tet resistance genes
Vancomycin Abnormal ligase and accessory genes
Mutations of acquired genes
Type of mutation Resulting gene / phenotype
Structural ESBL
Regulatory Mec (methicillin resistance) ESBL
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MUTATION
ENVIRONMENT
ADAPT
EVOLUTION
MAINTENANCE
STABILITY
FAITHFUL
GENERATION
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UV exposure
Ionizing radiation
Chemical exposure & Cellular metabolism
Oxidative deamination
Fidelity
Natural causes of point mutations
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STABILITY Ü Maintenance of genome stability
Ü Faithful copying over many generations
Ü Proof-reading (exonuclease activity of Pol III) Ü Mismatch repair systems
Mutations are random with respect to their effect on the fitness of the organism (host)
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Translation and the Genetic Code
Ü 61 sense codons and 3 stop codons
Ü 61 sense codons and 20 primary aa’s, 18/20 aa’s are encoded by >1 codon = degenerate code.
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Mutation Ü Substitution mutations
Ü Transitions (purine – purine eg A → G) Ü Transversions (pyrimidine → purine eg A → C) Ü Synonamous – silent mutations – no aa change (degenerate
code)
Ü Nonsynonamous – aa replacement Ü Missense Ü Nonsense
Ü 30% of all 3rd position changes are nonsynonamnous Ü 100% of all 2nd position changes are nonsynonamous Ü 96% of all 1st position changes are nonsynonamous
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Insertions / deletions
Recombination
Ü Homologous recombination Ü Site specific recombination
1. Unequal crossing over 2. Intrastrand deletion
n Site-specific rec when a repeated sequence pairs with another in the same orientation on the same chromatid
n Excision of a transposable element can involve recombination between direct repeats, 5 – 9 bp, flanking the element
3. Slipped-strand mispairing
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Amino acid Venn diagram
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Amino acid Series of amino acids – protein secondary structure
Tertiary protein structure
Surfaces, pockets, binding domains, conformational changes
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Mutations of preexisting genetic determinants
Type of mutation Resistance phenotype
Structural Streptomycin Rifampicin Fluoroquinolones Sulfonamides Trimethoprim
Regulatory Aminoglycosides (aarA) β-lactamases (AmpC) Chloramphenicol, Fluoroquinolones, tetracyclines (marA) Imipenem (OMP)
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NH
NNH
NNH2
O
OP
OP
OO
O
O
O
NH2 COO-
NH
NNH
N
O
NH2
CH2
NH
COO-
NH
NNH
NNH2
O
NH
NH
O
-O2CCOO-
NH
NH
NH
N
O
NH2
NH
NH
O
-O2C COO-
Dihydropterin pyrophosphate (DHPPP)
p-Aminobenzoate (pABA)
7, 8-Dihydropteroate (DHP)
Dihydrofolate (DHF)
Pyrophosphate
Dihydropteroate synthase (DHPS)
ATP + Glutamate
NADP
NADPH
Dihydrofolate reductase (DHFR)
Sulphonamide
+ DHP-Sulpha
Dihydrofolate synthase (DHFS)
Tetrahydrofolate (THF)
Trimethoprim
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Wild type
Mutant
Pneumocystis jirovecii DHPS
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Mutations of preexisting genetic determinants
Type of mutation Resistance phenotype
Structural Streptomycin Rifampicin Fluoroquinolones Sulfonamides Trimethoprim
Regulatory Aminoglycosides (aarA) β-lactamases (AmpC) Chloramphenicol, Fluoroquinolones, tetracyclines (marA) Imipenem (OMP)
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Group A
TEM & SHV
Pen Cef ’s
Penems
Inhibitor Sensitive
Group C
AmpC
Cef ’s Oxa
Inhibitor Resistant
Group D
OXA
Pen esp Oxa
Inhibitor
S / R
Group B
IMP & VIM
Penems
Inhibitor Resistant
Active site Serine Active site Zn
(metallo)
β-lactamase classification
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http://www.psc.edu/science/2006/enzyme
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Gram-positive
• Group A
Components
B. licheniformis
1. blaR1
2. blaR2
3. blaI
4. blaP
Gram-negative
• Group C
Components
C. fruendii
1. ampC
2. ampR
3. ampD
4. ampG
Bennett, PM. Antimicrob Agent Chemother 1993;37(2).
Gregory, PD. Mol Microbiol 1997;24(5).
Jacobs, C. Science 1997;278(5344).
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Inducible Gram-positive β-lactamase
blaR1 blaI O blaP
blaP blaI
blaR1
blaP blaP
blaP
blaR2
blaR2
β-lactams
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blaR1 blaI O blaP
blaP blaI
blaR1
blaR2
blaR2
Inducible Gram-positive β-lactamase
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blaR1 blaI O blaP
blaP blaI
blaR1
blaR2
blaR2
Noninducible basal-level expression of blaP
Inducible Gram-positive β-lactamase
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blaR1 blaI O blaP
blaP blaI
blaR1
blaP blaP
blaP
blaR2
blaR2
Constitutive high-level expression of blaP
Inducible Gram-positive β-lactamase
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Gram-negative
• The inducible β-lactamases are exclusively chromosomal genes
• AmpC – extended phylogenetically related family, some members are no longer inducible e.g. β-lactamases of E. coli, Shigella and Salmonella spp.
Inducible Gram-negative ampC β-lactamase
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Inducible Gram-negative ampC β-lactamase
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Inducible Gram-negative ampC β-lactamase
ampD Null mutant
Derepressed
Constitutive hyperproducer
ampD Hyperproducer
More sensitive to inducer
3 – 4X expression
ampR Non-inducible
2 – 3X expression
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Acquisition of foreign DNA
Phenotype Acquired genes
Aminoglycosides Aminoglycoside-modifying enzymes
β-lactams β-lactamase genes
Chloramphenicol CAT genes
Erythromycin/clindamycin Methylase/MLSB genes
Methicillin mecA gene
Penicillin PBP genes, β-lactamase
Sulfa DHPS gene
Trimethoprim DHFR gene
Tetracyclines Tet resistance genes
Vancomycin Abnormal ligase and accessory genes
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Bacteria have inhabited the earth for > 3.5 billion years, competing for survival, and evolving chemical defenses against
rival species – antibiotics.
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Some clinically important antibiotics
Antibiotic Producer organism Activity Site or mode of action
Penicillin Penicillium chrysogenum Gram-positive bacteria Wall synthesis
Cephalosporin Cephalosporium acremonium Broad spectrum Wall synthesis
Griseofulvin Penicillium griseofulvum Dermatophytic fungi Microtubules
Bacitracin Bacillus subtilis Gram-positive bacteria Wall synthesis
Polymyxin B Bacillus polymyxa Gram-negative bacteria Cell membrane
Amphotericin B Streptomyces nodosus Fungi Cell membrane
Erythromycin Streptomyces erythreus Gram-positive bacteria Protein synthesis
Neomycin Streptomyces fradiae Broad spectrum Protein synthesis
Streptomycin Streptomyces griseus Gram-negative bacteria Protein synthesis
Tetracycline Streptomyces rimosus Broad spectrum Protein synthesis
Vancomycin Streptomyces orientalis Gram-positive bacteria Protein synthesis
Gentamicin Micromonospora purpurea Broad spectrum Protein synthesis
Rifamycin Streptomyces mediterranei Tuberculosis Protein synthesis
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The other side of the coin however:
Many species of pro- and eukaryotes (especially fungi) have equally evolved counter measures against antibiotics for an equal amount of time.
Now an environmental library of resistance genes exist.
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Normal microbiota
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Artificial environments e.g. indwelling medical devices
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Ü Cattle 104 – 110 million
Ü Chickens 7.5 – 8.6 billion
Ü Turkey 275 – 292 million
Ü Swine 60 – 92 million
Ü Antibiotics used: 9.3 million Kg / year
Ü Meat producing animals excrete 1400 billion Kg waste / year
Agriculture & Veterinary
Sarmah, AK. Chemosphere. 2006;65.
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Central lending library: Mechanisms of acquiring foreign DNA
(Horizontal Gene Transfer)
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Genetic mechanisms of antibiotic resistance acquisition among common pathogenic bacteria
Mutation Natural transformation Conjugative transfer
All bacteria M. Tuberculosis
Acinetobacter Enterococcus Helicobacter Haemophilus Neisseria Staphylococcus Streptococcus
Enterobacteriacaea* Acinetobacter Campylobacter Bacteroides Clostridia Enterococcus Haemophilus Helicobacter Mycoplasma Listeria Neisseria Pseudomonas Staphylococcus Streptococcus Vibrio Yersinia
* Enterobacter, E. coli, Klebsiella, Proteus, Salmonella, Shigella, Serratia
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Hospitals: Convenient ecosystem for gene transfer
Ü Many patients Ü Continuous change Ü Reservoirs Ü Selective antibiotic
pressure
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Genetic pool and HGT “selfish gene”
Ü Ability to take up DNA Ü Willingness to deliver
DNA Ü In proximity at the same
time Ü Encountering free DNA
circulating in the environment
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Bacteriophages are bacterial viruses
Ü Major contributor to the evolution of bacteria
Ü DNA of phage origin often comprize 10 – 20% of bacterial genomes
Ü 2/3 of gamma proteobacteria harbor intact / remnant bacteriophage genomes
Ü Ubiquitous in GIT (107/g), marine and soil systems, and sewage
Transduction
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Ü Process by which bacteria take up naked (free) DNA from the environment
Ü Restrictions apply (restriction modification system)
Ü S. pneumoniae, viridans streptococci, H. influenzae, N. gonorrhoea,
N. meningitidis
Transformation
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Homologous recombination
Fate of incoming foreign DNA
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S. pneumoniae PBP2b
Wild-type
United Kingdom 1987
Chech Republic 1987
Papua New Guinea 1970
Kenya 1992
South Africa 1990
Papua New Guinea 1970 20%
4%
30%
21%
S. pneumoniae
S. mitis
S. oralis
Strep?
Strep?
Dowson, CG. Trends Microbiol. 1994;361.
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Dessen, A. J Biol Chem. 2001;276.
Structural comparison between Sp328 and R6 PBP2x*
Figure shows superposition of PBP2x* from Sp328 (green) and from the penicillin-sensitive R6 strain (blue). Most C chain divergences occur at the level of the N-terminal domain, which is much more stable and the 360–394 loop region (red), flexible in the penicillin-resistant molecule.
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Dessen, A. J Biol Chem. 2001;276
Drug-sensitive and -resistant active sites
A, active site of PBP2x* from penicillin-sensitive strain R6 (1QME.pdb). The three crucial motifs for enzymatic activity are represented by Ser337 (SXXK), Ser395 (SXN), and Lys547 (K(S/T)G). Thr338 is at the N-terminal end of 2, and Asn514 points away from 4, which harbors Ser389. B, active site of PBP2x* from penicillin-resistant strain Sp328. Although all three motifs are represented, the SXN loop is clearly displaced, probably the result of a steric clash between Leu389 and His514, which points into where 4 should be located. Ser347 not only adds a polar character to the region but also makes contact with Thr352, a residue present in the loop that follows 2 (not shown for clarity).
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Conjugation
Ü Method by which bacterial cells come into contact with each other to exchange genetic material
Ü Machinery required for conjugation (pilus and transfer) encodes by a plasmid in the donor
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Plasmids are extra-chromosomal circular DNA. Encodes accessory functions including antimicrobial resistance, carbohydrate fermentation, bacteriocins, toxins, adhesive and colonization factors, conjugation
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Natural history of emergence of resistance to β-lactams and aminoglycosides
Conjugative transfer
Gram-positive Gram-negative
Soil microorganisms
1965 β-lactamase S. aureus 1965 β-lactamase E. coli
1970 β-lactamase & aminoglycosides S. aureus
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Courvalin P, Antimicrob Agent Chemother. 1994;38.
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Plasmids
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Ü Integrons are elements containing the genetic determinants of the components of a site-specific recombination system that recognizes and captures mobile gene cassettes.
Ü Integrase (int) and adjacent recombination sites (attI).
Ü Gene cassette consist of one coding sequence
Integrons & gene cassettes
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Although integrons themselves are not mobile, they are sometimes found as part of transposons. These transposons are generally located on plasmids – further enhancing their spread.
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Fluit, AC. Eur J Microbiol Infect Dis. 1999;18.
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Integrons abound
• France: 59% in Enterobacteriaceae from clinical specimens (n=49).
• Germany: 13% in 11 Gram-negative species from blood cultures (n=278).
• Nine countries: 42% in 13 species of Gram-negative from clinical specimens (n=163).
• Integrons in staphylococci and enterococci
• Integrons from primates Fluit, AC. Eur J Microbiol Infect Dis. 1999;18.
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Chromosome
Conjugative Plasmid
tra1
oriT
tra2
Transposon
conjugation TetM Van β-lac
Rec
vanR vanW vanY vanB
Integron
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Mutation of acquired genes: Structural, β-lactamases
Mutations of acquired genes
Type of mutation Resulting gene / phenotype
Structural ESBL Regulatory Mec (methicillin resistance)
ESBL
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Mutation SHV β-lactamase
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Group A
TEM & SHV
Pen Cef ’s
Penems
Inhibitor Sensitive
Group C
AmpC
Cef ’s Oxa
Inhibitor Resistant
Group D
OXA
Pen esp Oxa
Inhibitor
S / R
Group B
IMP & VIM
Penems
Inhibitor Resistant
Active site Serine Active site Zn
(metallo)
β-lactamase classification
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Evolution of Class A SHV β-lactamases
Ü Functionality
Ü Enzyme active-site residues
Ü 3-D conformation
Ü Expanding substrate range
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HSV-2 HSV-5 HSV-8 HSV-9
HSV-4
HSV-12
HSV-1 HSV-10
HSV-11 HSV-2a
HSV-3 HSV-6 ? HSV-7
238
8 35 43 54 130 140 179 192 193 195 205 238 240
SHV-1 Klebsiella 1974 I L R G S A D K L T R G E Pen
SHV-2 Klebsiella, Serratia 1983 S ESBL
SHV-2a Klebsiella 1990 Q S ESBL
SHV-3 Klebsiella 1988 L S ESBL
SHV-4 Klebsiella 1988 L S K ESBL
SHV-5 Klebsiella 1989 S K ESBL
SHV-6 Klebsiella 1991 A CAZ
SHV-7 Escherichia 1995 F S S K ESBL
SHV-8 Escherichia 1997 N ESBL
SHV-9Eschirichia, Klebsiella,
Serratia1995 Del R N V S K ESBL
SHV-10 Eschirichia 1997 Del G R N V S K IR ESBL
SHV-11 Enterobacteriaceae 1997 Q Pen
SHV-12 Enterobacteriaceae 1997 Q S K ESBL
SpectrumAmino acid at positionß-
LactamaseOrigin Country Year
205
240
240
205 179 43
8
179 ?
54, 140
192, 193
130
35 35
238
35
?
?
Heritage, J. J Antimicrob Chemother. 1999;44.
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Clonal Non clonal
(gene exchange)
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Nosocomial spread of resistance plasmid
Ü Outbreak of ESBL and aminoglycoside-resistant E. cloacae (June – November 2000) followed by…
Ü Isolation of ESBL and aminoglycoside-resistant A. baumanii (November 2000)
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E. cloacae
A. baumanii
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Al Naiemi, N. J Clin Microbiol 2005:43
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Ü Infection control measures after E. cloacae outbreak Ü Hand hygiene Ü Gloves and gowns during patient care activities Ü Isolation of patients with MDR E. cloacae infections in
private rooms Ü These measures failed to prevent the clonal MDR A. baumanii infections
Ü Other Gram-negative bacteria may have acted as a reservoir of the plasmid
Ü Infection control measures after A. baumanii infections Ü Control measures to all patients in ICU Ü Closure of ICU to new admissions Ü Dedicated nursing team for patients colonized with the resistant strain
Ü These measures were successful in halting the MDR A. baumanii outbreak
Ü Lead to a significant decrease of all MDR-Gram-negative bacilli Al Naiemi, N. J Clin Microbiol 2005:43
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