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Transcript of 12 August 2003 AmpC -Lactamases & their detection David Livermore Health Protection Agency,...
12 August 2003
AmpC -Lactamases& their detection
David Livermore
Health Protection Agency,
Colindale, London
-Lactamase-stable cephs
CONH
O
COOH
R
C
NOR
H2N
Oxyimino-aminothiazolylstability to classical TEM/SHV
S NS
N
-Lactamase classes
Chromosomal Plasmid
A Bacteroides,Klebsiella,P. vulgaris
Staph pen’aseTEM, SHV
B S. maltophilia,flavobacteria
IMP, VIM
C Mostenterobacteria
CMY, LAT, FOX
D Aeromonas OXA
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
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
[ -lactam]
Am
t -
lact
am
ase
Derepressed
Inducible
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
Uninduced AmpC
• Wall fragments recycled by AmpD
• AmpR in repressor conformation
• ampC (-lactamase gene) NOT expressed
ampCampR
ampD
AmpD AmpR
Induced AmpC
ampCampR
ampD
• More recycling: AmpD overwhelmed
• Wall fragments convert AmpR to activator
• ampC (-lactamase gene) expressed
AmpD-lactamase
Derepressed AmpC
ampCampR
ampD
• ampD inactivated by mutation
• AmpR constantly converted to activator
• ampC hyper-expressed
-lactamase++
Strong & weak inducers
Strong inducer Weak inducer
Labile 1st gen cephs
Ampicillin
3rd gen cephs
Piperacillin
Aztreonam
Stable Imipenem (Temocillin)
(Meropenem)
Strong inducer induces below MIC, weak doesn’t
MICs (mg/L) for E. cloacaeAmpC mutants
Inducible Derepressed Basal Ampicillin 512 2048 4 Cephalothin 256 1024 16 Piperacillin 4 128 1 Cefotaxime 0.5 256 0.06 Ceftazidime 0.25 256 0.25 Aztreonam 0.06 16 0.06 Imipenem 0.25 0.25 0.06 Meropenem 0.06 0.12 0.015
MICs (mg/L) for P. aeruginosa AmpC mutants
Inducible SDR Basal
Ampicillin 512 2048 32
Carbenicillin 32 64 32
Piperacillin 2 128 2
Ceftazidime 2 32 2
Cefepime 4 32 2
Imipenem 2 2 0.25
Meropenem 0.25 0.25 0.25
Strong inducers –e.g. imipenem
What selects derepression?
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
Over-run by mutants
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...
Mutant emerges randomly
Sensitivecells killedby antibiotic
Mutant’s progeny overrun
Initial isolation of 3rd-gen cephR Enterobacter, & prior Rx
Prior Rx Incidence %
Any antibiotic 36/103 35
No antibiotic 1/26 4
3rd gen ceph 22/32 69
No 3rd gen ceph 14/71 20
Chow et al. Ann Intern Med 1991, 115, 585-90
Selecting Enterobacter R to 3rd gen cephs during Rx
Case ControlMortality 26% 13%Mean stay 29.5 days 19 daysMedian cost $79,000 $40,000
Selection by cephsOverall 19%Bacteraemia 29%Tissue, urine, wounds 7%
Kaye et al. AAC 2001, 45, 2628; Cosgrove et al. Arch Intern Med 2002, 162, 185
Plasmidic AmpC enzymes
Escaping from enterobacterial and Aeromonas chromosomes
Many good reports since 1991
CMY, MOX, FOX, LAT, DHA, ACT & BIL enzymes
Sources of plasmid AmpC
Class Source Examples
CIT C. freundii CMY-2 to 7; LAT-1,3,4
ENT Enterobacter spp. ACT-1; MIR-1
FOX Aeromonas spp. FOX-1 to -5;
MOX Aeromonas spp. MOX-1,-2; CMY-1,7 & 8
DHA M morganii DHA-1, -2
ACC H. alvei ACC-1
Plasmid AmpC
MIC (mg/L)
K. pneum 77 E. coli R- E. coli R+
Ampicillin 512 4 512
Co-amoxiclav 256 2 512
Cefotaxime 8 0.03 8
Ceftazidime 8 0.06 16
Ctaz + clav 8 0.06 16
Cefoxitin 32 4 16
Meropenem 0.06 0.06 0.25
-Lactamases TEM-1, SHV-1 AmpC
TEM-1, AmpC
Jenks et al. 1995. J Antimicrob Chemother 35, 235-6.
Enzymes in 1127 cephR isolates from 16 labs in S England, 2004
0
100
200
300
400
500
600
E. coli K. pneumoniae E. cloacae
CTX-M Other ESBL AmpC Other
Potz et al., JAC, 2006 in press
Prevalence of mechanisms: BSAC bacteraemia surveillance, 2005
0%
20%
40%
60%
80%
100%
E. coli Klebsiella Enterobacter
K1BothESBLAmpCS
http://www.bsacsurv.org
Inducible AmpC
Cefoxitin
Ceftazidime
But mutational derepression is the problem, not induction
Better predict risk from species I/D
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
Geometric mean MICs (mg/L): AmpC producers; 2004 London SE survey
E. coli AmpC
E. coli ESBL
Enterobacter AmpC
Enterobacter ESBL
Cefotaxime 12 228 72 49
Ceftazidime 18 39 36 81
Cefepime 0.38 39 0.91 4.4
Cefpirome 0.57 53 2.4 10
Cefuroxime 35 >64 >64 >64
Cefoxitin 51 17 >64 51
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 there’s an ESBL with the AmpC
Confirmatory tests for AmpC
• Seek cefotaxime/cloxacillin synergy
–Cefotaxime MIC +100 mg/l cloxacillin
–Zones of cefotaxime 30 g 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
Cefotaxime combinations vs. AmpC E. coli: London SE survey
0
5
10
15
20
25
30
<=0.
120.
25 0.5 1 2 4 8 16 32 64 >64
MIC (mg/L)
Alone
+Clavulanate, 4 mg/L
+Cloxacillin 100 mg/L
Cefotaxime combinations vs. AmpC E. coli: London SE survey
0
10
20
30
40
50
60
70
<=0.
120.
25 0.5 1 2 4 8 16 32 64 >64
MIC (mg/L)
Alone
+Clavulanate, 4 mg/L
+Cloxacillin 100 mg/L
Cefotaxime / cloxacillin tests for AmpC
1
10
100
1000
E. cloacae 684-con P. aeruginosa 2297-con
MIC
(m
g/L
)
Cefotaxime
Cefotaxime +cloxacillin
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
Clover leaf (3 dimensional) test for AmpC
Test strainE. cloacae,
AmpC derepressed
IndicatorE. coli
NCTC10418
DiscCefoxitin 30 g
Clover leaf (3 dimensional) test for cephalosporinase
Test strainE. cloacae,
AmpC derepressed
IndicatorE. coli
NCTC10418
DiscCefotaxime 30 g
Phenyl boronic acid for detection of plasmid AmpC
Expansion (mm) of FOX 30 zone with 400 g phenyl boronic acid
Fold MIC reduction for cefoxitin + 400 mg/L phenyl boronic acid
Kleb MOX-1 12 128
E. coli LAT-2 12 64
Kleb DHA-1 14 128
Kleb DHA-2 13 64
E. coli ACC-1 4 4
Kleb ACT-1 13 64
ALL ESBL +ve <2 <2
Coudron JCM 2005 43 4163
Disc tests for AmpC
60 E. coli & Klebsiella : cefoxitin MICs reduced >4-fold by 100 mg/L cloxacillin
% with >5 mm zone expansion
Cefoxitin + cloxacillin 100 g 86%
Cefoxitin + BZB 64 g 89%
Cefpodoxime + BZB 64 g 97%
Cefpodoxime + clav + BZB 64 g 100%
BZB: benzo(b)thiophene-2-boronic acid
Brenwald et al., JAC 2005, 56, 600
Cefoxitin R isolates in phenyl boronic acid / cefoxitin tests
3-D Phenyl boronic, disc
Phenyl boronic MIC
+ - + - + -
K. pneumoniae 106 16 110 12 110 12
K. oxytoca 4 2 5 1 5 1
E. coli 22 93 40 75 39 76
P. mirabilis 24 4 25 3 26 2
Coudron JCM 2005 43 4163
AmpC hyperproducers : options
Active
Carbapenems
Temocillin
4-gen cephs
Not active
Penicillins except temocillin
Inhibitor combinations
1, 2, 3-gen cephs
Aztreonam
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