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  • ß-lactamases Clinical Impact and Epidemiology

    Professor Sebastian Amyes

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

    β-lactamases Clinical Impact and Epidemiology

    1

    Professor Sebastian Amyes University of Edinburgh

    Edinburgh Scotland

    β-lactamases in Gram-negative bacteria

    >750 β-lactamases reported

    …of clinical importance

    Cephalosporins

    2

    Transferable class C β-lactamases Extended-spectrum β-lactamases (ESBLs)

    Carbapenems Transferable class B carbapenamses Transferable class D carbapenamses

    β-lactamase action

    1. HO

    H N H

    N

    SN

    O O

    2

    N

    S

    H

    3

    Class & active site

    Associated substrate

    A Serine

    B Metal ion

    C Serine

    D Serine

    Penicillin Carbapenem Cephalosporin Penicillin

    COO-

    β-lactamase

    COO-H

  • ß-lactamases Clinical Impact and Epidemiology

    Professor Sebastian Amyes

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

    1. Induction Interference with repressor protein

    2 De-repression

    Enterobacter spp Chromosomal β-lactamase expression

    Chromosome

    4

    2. De repression Mutation in R gene

    P Rβ-lactamase

    Repressor

    Emergence of the BIL-1 β-lactamase Mobilisation of chromosomal class C genes onto plasmids

    Chromosome Plasmid

    Resistant Enterobacteriaceae

    ChromosomePlasmid

    Klebsiella spp or Escherichia coli

    5

    P Rβ-lactamase

    Dendrogram for AmpC β-lactamases plasmid and chromsomal

    E. coli K12

    Ent. cloacae ACT-1

    MIR-1

    Ent. aerogenes

    Citrobacter sppA. baumannii RYC

    P. immobilis A5

    M. morganii Y. enterocolitica IP97

    DHA-2 DHA-1 GN7471

    1113

    BIL-1 CMY-4 CMY-7 CMY-3 CMY-6 LAT-4 LAT-3 CMY-5 LAT-1

    GN346

    P99

    6Data from: Philippon et al., (2002). Antimicrob. Agents Chemother. 46:1

    Aeromonas spp

    P. aeurginosa PAO1

    O. anthropi P. stuartii VDG96

    H. alvei

    L. lactamgenus YK90

    SLH62 ACC-1

    SLS73 SST-1 SRT-1 82116

    2171

    MOX-1 MOX-2

    CMY-1 CMY-8 CMY-9

    CepS - A. sobria

    CepH - A. hydrophila

    AER14 - A. sobria

    FOX-3 FOX-4 FOX-2 FOX-5 FOX-1

    S. marcescens

  • ß-lactamases Clinical Impact and Epidemiology

    Professor Sebastian Amyes

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

    Transferable AmpC β-lactamases CMY ACC ACT FOX MIR MOX Others CMY-1 ACC-1 ACT-1 FOX-1 MIR-1 MOX-1 CFE-1 CMY-2 ACC-2 ACT-2 FOX-2 MIR-2 MOX-2 CMY-3 ACC-3 ACT-3 FOX-3 MIR-3 MOX-3 DHA-1 CMY-4 ACC-4 ACT-4 FOX-4 MIR-4 MOX-4 DHA-2 CMY-5 ACT-5 FOX-5 MIR-5 MOX-5 CMY-6 ACT-6 FOX-6 MOX-6 LAT-1 CMY-7 ACT-7 FOX-7 MOX-7 CMY-8 CMY-9 CMY-10 CMY-11 CMY-12 CMY 13

    7Total ~ 78

    CMY-13 CMY-14 CMY-15 CMY-16 CMY-17 CMY-18 CMY-19 CMY-20 CMY-21 CMY-22

    ¦ ¦ ¦

    CMY-44

    ~44 4 ~7 7 5 7 4

    What are extended-spectrum β-lactamases?

    “Extended-Spectrum β-lactamases (ESBLs) are enzymes that can be produced by bacteria making them resistant to cephalosporins”

    8

    making them resistant to cephalosporins

    Health Protection Agency

    http://www.hpa.org.uk/infections/topics_az/esbl/default.htm

    OXA 1

    TEM-2

    SHV-1

    Dominance of the TEM-1 β-lactamase

    Accounts for 75% of all plasmid-encoded enzymes

    9

    OXA-1

    PSE-1 CARB-6

    TEM-1 OXA-2

    SAR-2

  • ß-lactamases Clinical Impact and Epidemiology

    Professor Sebastian Amyes

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

    What happened in Liverpool in 1982?

    TEM-1

    Extensive ceftazidime use

    10

    TEM-1 TEM-1

    Gene duplication – small increase in MIC

    TEM-1 TEM-12

    Mutation at codon 164 – MIC increases to 4mg/L

    ß-3 ß-4

    α-10 α-2

    α-9

    Binding of amoxycillin

    Active site of the TEM-1 β-lactamase

    11

    α-7

    α-3

    α-5

    α-4

    Ω-loop

    Ser-70

    Asp-179

    Arg-164

    Glu-171

    ß-3 ß-4

    α-10 α-2

    α-9

    Active site of the TEM-1 β-lactamase Failure to bind cephalosporins

    12

    α-7

    α-3

    α-5

    α-4 Ser-70

    Arg-164

    Asp-179

    Glu-171 Ω-loop

  • ß-lactamases Clinical Impact and Epidemiology

    Professor Sebastian Amyes

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

    ß-3 ß-4

    α-10 α -2

    α-9

    Serine for arginine substitution at 164 (TEM-12)

    13

    α-7

    α-3

    α-5

    α-4 Ser-70

    Ser-164

    Asp-179

    Glu-171

    Amino acid substitutions in TEM ESBLs

    Leu 21

    Gln 39

    Ala 42

    Leu 51

    Gly 92

    Glu 104

    His 153

    Arg 164

    Met 182

    Gly 218

    Ala 237

    Gly 238

    Glu 240

    Arg 244

    Thr 265

    Ser 268

    Phe TEM-4 TEM-9 TEM-25 TEM-48 TEM-49 TEM-53 TEM-63

    Val TEM-42

    Asp TEM-57 TEM-66

    Arg TEM-21 TEM-56

    Thr TEM-20 TEM-43 TEM-52 TEM-63 TEM-72

    Lys TEM-5 TEM-10 TEM-24 TEM-27 TEM-28 TEM-42 TEM-46 TEM-47 TEM-48

    TEM-49 TEM-61 TEM-68 TEM-72

    Ser TEM-50Thr

    TEM-5 TEM-24

    Gly TEM-22

    Met TEM-4 TEM-9 TEM-13 TEM-25 TEM-27 TEM-42 TEM-47 TEM-48 TEM-49 TEM-68

    TEM-1

    14

    Lys TEM-3 TEM-4 TEM-6 TEM-8 TEM-9 TEM-14 TEM-15 TEM-16 TEM-17 TEM-18 TEM-21

    TEM-22 TEM-24 TEM-26 TEM-43 TEM-46 TEM-50 TEM-52 TEM-56 TEM-60 TEM-63 TEM-66

    Lys TEM-2 TEM-3 TEM-7 TEM-8 TEM-11 TEM-13 TEM-16 TEM-18 TEM-21 TEM-22 TEM-42 TEM-46 TEM-56 TEM-60 TEM-61 TEM-66 TEM-72

    Pro TEM-60

    Glu TEM-55

    Ser TEM-5 TEM-7 TEM-8 TEM-9 TEM-10 TEM-12 TEM-22 TEM-25 TEM-26 TEM-46 TEM-53 TEM-60 TEM-63

    His TEM-6 TEM-11 TEM-16 TEM-27 TEM-28 TEM-29 TEM-43 TEM-61

    Ser TEM-3 TEM-4 TEM-8 TEM-14 TEM-15 TEM-18 TEM-19 TEM-20 TEM-21 TEM-22 TEM-25 TEM-42 TEM-47 TEM-48 TEM-49 TEM-52 TEM-66 TEM-68 TEM-72

    Leu TEM-54

    Ser TEM-58

    Gly TEM-49

    Data from: Bradford (2001). Clin. Microbiol. Rev. 14:933

    Sequential mutation of ESBLs by cephalosporins

    3rd generation cephalosporins

    R164S E240K A237T

    15

    TEM-12 TEM-10 TEM-5TEM-1

    MIC (mg/l)

    Ceftazidime

  • ß-lactamases Clinical Impact and Epidemiology

    Professor Sebastian Amyes

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

    Porin

    Outside cell

    Outer membrane

    Interaction of cephalosporins with β-lactamases in Gram-negative bacteria

    Ceftazidime

    Porin

    Ceftriaxone or Cefotaxime

    16

    Penicillin binding proteinsβ-lactamase

    Periplasmic space

    Cytoplasm β-lactamase Penicillin binding proteins

    Progression of TEM-1 to TEM-5 β-lactamase Effect on hydrolysis rate and inhibitor sensitivity

    0.01

    0.1

    1

    10

    100

    s)

    Ceftazidime Cefotaxime Ampicillin Clavulanic acid

    17Data from Du Bois, Marriott & Amyes (1995). J. Antimicrob. Chemother. 35: 7 TEM-1 TEM-12 TEM-10 TEM-5

    1

    10

    100

    1000 0.001

    1000

    100

    10

    1

    K ca

    t (1

    /s

    C la

    vu la

    ni c

    ac id

    I 50 (n

    M )

    ß-3 ß-4

    α-10 α-2

    α-9

    Substitutions of the TEM-1 β-lactamase (ESBL formation)

    18 α-7

    α-3

    α-5

    α-4

    Ω-loop

    Ser-70

    Arg-164

    Ala-237

    Gly-238

    Glu-240Glu-104

  • ß-lactamases Clinical Impact and Epidemiology

    Professor Sebastian Amyes

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

    Lys SerLys Pro GluSer His Leu Gly

    Amino acid substitutions in TEM ESBLs

    Leu 21

    Gln 39

    Ala 42

    Leu 51

    Gly 92

    Glu 104

    His 153

    Arg 164

    Met 182

    Gly 218

    Ala 237

    Gly 238

    Glu 240

    Arg 244

    Thr 265

    Ser 268

    Phe TEM-4 TEM-9 TEM-25 TEM-48 TEM-49 TEM-53 TEM-63

    Val TEM-42

    Asp TEM-57 TEM-66

    Arg TEM-21 TEM-56

    Thr TEM-20 TEM-43 TEM-52 TEM-63 TEM-72

    Lys TEM-5 TEM-10 TEM-24 TEM-27 TEM-28 TEM-42 TEM-46 TEM-47 TEM-48

    TEM-49 TEM-61 TEM-68 TEM-72

    Ser TEM-50Thr

    TEM-5 TEM-24

    Gly TEM-22

    Met TEM-4 TEM-9 TEM-13 TEM-25 TEM-27 TEM-42 TEM-47 TEM-48 TEM-49 TEM-68

    TEM-1

    19

    Lys TEM-3 TEM-4 TEM-6 TEM-8 TEM-9 TEM-14 TEM-15 TEM-16 TEM-17 TEM-18 TEM-21

    TEM-22 TEM-24 TEM-26 TEM-43 TEM-46 TEM-50 TEM-52 TEM-56 TEM-60 TEM-63 TEM-66

    Ser TEM-3 TEM-4 TEM-8 TEM-14 TEM-15 TEM-18 TEM-19 TEM-20 TEM-21 TEM-22 TEM-25 TEM-42 TEM-47 TEM-48 TEM-49 TEM-52 TEM-66 TEM-68 TEM-72

    Lys TEM-2 TEM-3 TEM-7 TEM-8 TEM-11 TEM-13 TEM-16 TEM-18 TEM-21 TEM-22 TEM-42 TEM-46 TEM-56 TEM-60 TEM-61 TEM-66 TEM-72

    Pro TEM-60

    Glu TEM-55

    Ser TEM-5 TEM-7 TEM-8 TEM-9 TEM-10 T