AmpC bLactamases

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AmpC b-Lactamases their detection

• David Livermore
• Health Protection Agency,
• Colindale, London

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b-Lactamase-stable cephs
S
CONH
N
O
R
COOH
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b-Lactamase classes
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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

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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
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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

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Uninduced AmpC

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

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Induced AmpC
?-lactamase
AmpD
ampC
ampR
ampD

• More recycling AmpD overwhelmed
• Wall fragments convert AmpR to activator
• ampC (?-lactamase gene) expressed

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Derepressed AmpC
?-lactamase
ampC
ampR
ampD

• ampD inactivated by mutation
• AmpR constantly converted to activator
• ampC hyper-expressed

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Strong weak inducers
Strong inducer induces below MIC, weak doesnt
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MICs (mg/L) for E. cloacaeAmpC mutants
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MICs (mg/L) for P. aeruginosa AmpC mutants
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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
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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
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Initial isolation of 3rd-gen cephR Enterobacter,
prior Rx
Chow et al. Ann Intern Med 1991, 115, 585-90
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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
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Plasmidic AmpC enzymes

• Escaping from enterobacterial and Aeromonas
  chromosomes
• Many good reports since 1991
• CMY, MOX, FOX, LAT, DHA, ACT BIL enzymes

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Sources of plasmid AmpC
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Plasmid AmpC
Jenks et al. 1995. J Antimicrob Chemother 35,
235-6.
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Enzymes in 1127 cephR isolates from 16 labs in S
England, 2004
Potz et al., JAC, 2006 in press
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Prevalence of mechanisms BSAC bacteraemia
surveillance, 2005
http//www.bsacsurv.org
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Inducible AmpC
Cefoxitin
But mutational derepression is the problem, not
induction Better predict risk from species I/D
Ceftazidime
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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

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Geometric mean MICs (mg/L) AmpC producers 2004
London SE survey
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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

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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

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Cefotaxime combinations vs. AmpC E. coli London
SE survey
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Cefotaxime combinations vs. AmpC E. coli London
SE survey
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Cefotaxime / cloxacillin tests for AmpC
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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
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Clover leaf (3 dimensional) test for AmpC
Test strain E. cloacae, AmpC derepressed Indicato
r E. coli NCTC10418 Disc Cefoxitin 30 mg
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Clover leaf (3 dimensional) test for
cephalosporinase
Test strain E. cloacae, AmpC derepressed Indicato
r E. coli NCTC10418 Disc Cefotaxime 30 mg
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Phenyl boronic acid for detection of plasmid AmpC
Coudron JCM 2005 43 4163
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Disc tests for AmpC
BZB benzo(b)thiophene-2-boronic acid
Brenwald et al., JAC 2005, 56, 600
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Cefoxitin R isolates in phenyl boronic acid /
cefoxitin tests
Coudron JCM 2005 43 4163
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Multiplex detection of plasmid AmpC genes
Method of Perez-Perez Hanson JCM 2002, 40, 2153
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AmpC hyperproducers options

• Active
• Carbapenems
• Temocillin
• 4-gen cephs

• Not active
• Penicillins except temocillin
• Inhibitor combinations
• 1, 2, 3-gen cephs
• Aztreonam

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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