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AmpC bLactamases

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12 August 2003. AmpC b-Lactamases & their detection. David Livermore ... c. 40,000; alkaline pI. Hydrolytic activity. 1st gen cephs rapid. 2/3 ceph cephs slow ... – PowerPoint PPT presentation

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Title: AmpC bLactamases


1
AmpC b-Lactamases their detection
  • David Livermore
  • Health Protection Agency,
  • Colindale, London

2
b-Lactamase-stable cephs
S
CONH
N
O
R
COOH
3
b-Lactamase classes
4
AmpC enzymes
  • Mol wt. c. 40,000 alkaline pI
  • Hydrolytic activity
  • 1st gen cephs rapid
  • 2/3 ceph cephs slow but kinetically efficient
  • 4-gen cephs slow, kinetically inefficient
  • Carbapenems nearly stable. not quite!
  • Not inhibited by clavulanate poorly inhibited by
    sulphones

5
AmpC ?-lactamases
  • Basal in
  • E. coli shigellae
  • Inducible in
  • Enterobacter spp.
  • C. freundii
  • M. morganii
  • Serratia spp.
  • P. aeruginosa
  • 2nd, 3rd gen cephs
  • Labile, but weak inducers, select derepressed
    mutants

Derepressed
Inducible
Amt ?-lactamase
? -lactam
6
Induction of AmpC
  • Cell wall constantly re-cycled
  • Yields disaccharide tri-peptides (DTPs)
  • Absorbed by AmpG
  • Cleaved by AmpD N-acetyl-muramyl-L-alanine
    amidase
  • Excess DTPs bind AmpR, activating ampC

7
Uninduced AmpC
  • Wall fragments recycled by AmpD
  • AmpR in repressor conformation
  • ampC (?-lactamase gene) NOT expressed

8
Induced AmpC
?-lactamase
AmpD
ampC
ampR
ampD
  • More recycling AmpD overwhelmed
  • Wall fragments convert AmpR to activator
  • ampC (?-lactamase gene) expressed

9
Derepressed AmpC
?-lactamase
ampC
ampR
ampD
  • ampD inactivated by mutation
  • AmpR constantly converted to activator
  • ampC hyper-expressed

10
Strong weak inducers
Strong inducer induces below MIC, weak doesnt
11
MICs (mg/L) for E. cloacaeAmpC mutants
12
MICs (mg/L) for P. aeruginosa AmpC mutants
13
What selects derepression?
  • Strong inducers e.g. imipenem

No! derepressed no more R than inducible
All weak inducers?
No- Not if they are stable
LABILE weak inducers?
Yes! Derepressed are R, whilst inducible cells
are S, so derepressed are selected
14
Over-run by mutants
Mutant emerges randomly
  • Derepression occurs in 1 cell / 107
  • Confers resistance to 3-gen cephs
  • Overnight, one cell can give 109 progeny
  • Selection in therapy can cause Rx failure...

Sensitive cells killed by antibiotic
Mutants progeny overrun
15
Initial isolation of 3rd-gen cephR Enterobacter,
prior Rx
Chow et al. Ann Intern Med 1991, 115, 585-90
16
Selecting Enterobacter R to 3rd gen cephs during
Rx
Kaye et al. AAC 2001, 45, 2628 Cosgrove et al.
Arch Intern Med 2002, 162, 185
17
Plasmidic AmpC enzymes
  • Escaping from enterobacterial and Aeromonas
    chromosomes
  • Many good reports since 1991
  • CMY, MOX, FOX, LAT, DHA, ACT BIL enzymes

18
Sources of plasmid AmpC
19
Plasmid AmpC
Jenks et al. 1995. J Antimicrob Chemother 35,
235-6.
20
Enzymes in 1127 cephR isolates from 16 labs in S
England, 2004
Potz et al., JAC, 2006 in press
21
Prevalence of mechanisms BSAC bacteraemia
surveillance, 2005
http//www.bsacsurv.org
22
Inducible AmpC
Cefoxitin
But mutational derepression is the problem, not
induction Better predict risk from species I/D
Ceftazidime
23
Suspect derepressed / plasmid AmpC if
  • Resistant 3-gen cephs, NOT cefepime cefpirome
  • Resistant to cefoxitin (but more ESBL producers
    R, too, nowadays)
  • No ceph/clav synergy

24
Geometric mean MICs (mg/L) AmpC producers 2004
London SE survey
25
Some wrinkles
  • AmpC-derepressed M. morganii are S to pip/tazo
  • AmpC derepressed Serratia are S to ceftazidime
  • Cefoxitin R an unreliable marker for Providencia,
    Morganella Serratia spp.
  • Inducible derepressed strains may appear I or S
  • AmpC derepressed P. aeruginosa tend to be S to
    carbenicillin / efflux mutants are R
  • Life complicated if theres an ESBL with the AmpC

26
Confirmatory tests for AmpC
  • Seek cefotaxime/cloxacillin synergy
  • Cefotaxime MIC 100 mg/l cloxacillin
  • Zones of cefotaxime 30 mg discs on agar 100
    mg/L cloxacillin
  • No agreed interpretive standards
  • Can also use phenylboronic acid as inhibitor
  • DHA enzymes inducible especially difficult to
    detect

27
Cefotaxime combinations vs. AmpC E. coli London
SE survey
28
Cefotaxime combinations vs. AmpC E. coli London
SE survey
29
Cefotaxime / cloxacillin tests for AmpC
30
3-D test for AmpCs
Plate seeded with cefoxitin S indicator strain
Cut cross in agar, heavily inoculated with test
strain
Cefoxitin disc
Looks for distortion where cross intersects the
cefoxitin zone
31
Clover leaf (3 dimensional) test for AmpC
Test strain E. cloacae, AmpC derepressed Indicato
r E. coli NCTC10418 Disc Cefoxitin 30 mg
32
Clover leaf (3 dimensional) test for
cephalosporinase
Test strain E. cloacae, AmpC derepressed Indicato
r E. coli NCTC10418 Disc Cefotaxime 30 mg
33
Phenyl boronic acid for detection of plasmid AmpC
Coudron JCM 2005 43 4163
34
Disc tests for AmpC
BZB benzo(b)thiophene-2-boronic acid
Brenwald et al., JAC 2005, 56, 600
35
Cefoxitin R isolates in phenyl boronic acid /
cefoxitin tests
Coudron JCM 2005 43 4163
36
Multiplex detection of plasmid AmpC genes
Method of Perez-Perez Hanson JCM 2002, 40, 2153
37
AmpC hyperproducers options
  • Active
  • Carbapenems
  • Temocillin
  • 4-gen cephs
  • Not active
  • Penicillins except temocillin
  • Inhibitor combinations
  • 1, 2, 3-gen cephs
  • Aztreonam

38
AmpC Summary
  • Mutant selection the problem, not induction
  • Selection mostly in therapy with 3-gen cephs
  • AmpC types spreading to plasmids
  • Suspect AmpC if
  • R 3rd, not 4th gen cephs cefoxitin R, no
    ceph/clav synergy
  • Confirmatory tests poorly standardised, exploit
    synergy with cloxacillin or phenyl boronic acids
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