Transcript Title of presentation

AmpC b-Lactamases
& their detection
David Livermore
Health Protection Agency,
Colindale, London
12 August 2003
b-Lactamase-stable cephs
H2N
S
N
S
C
CONH
N
N
OR
O
Oxyimino-aminothiazolyl
stability to classical TEM/SHV
R
COOH
b-Lactamase classes
A
B
C
D
Chromosomal
Plasmid
Bacteroides,
Klebsiella,
P. vulgaris
S. maltophilia,
flavobacteria
Most
enterobacteria
Aeromonas
Staph pen’ase
TEM, SHV
IMP, VIM
CMY, LAT, FOX
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 b-lactamases
Basal in:
Derepressed
Inducible in:
Enterobacter spp.
C. freundii
M. morganii
Serratia spp.
P. aeruginosa
Amt b-lactamase
E. coli & shigellae
Inducible
2nd, 3rd gen cephs:
Labile, but weak inducers,
select derepressed mutants
[b -lactam]
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
AmpD
AmpR
ampD
amp
ampC
R
• Wall fragments recycled by AmpD
• AmpR in repressor conformation
• ampC (b-lactamase gene) NOT expressed
Induced AmpC
b-lactamase
AmpD
ampD
amp
R
ampC
• More recycling: AmpD overwhelmed
• Wall fragments convert AmpR to activator
• ampC (b-lactamase gene) expressed
Derepressed AmpC
b-lactamase++
ampD
amp
R
ampC
• ampD inactivated by mutation
• AmpR constantly converted to activator
• ampC hyper-expressed
Strong & weak inducers
Strong inducer Weak inducer
Labile
Stable
1st gen cephs
3rd gen cephs
Ampicillin
Piperacillin
Imipenem
Aztreonam
(Temocillin)
(Meropenem)
Strong inducer induces below MIC, weak doesn’t
MICs (mg/L) for E. cloacae
AmpC mutants
Inducible Derepressed Basal
512
2048
4
Ampicillin
256
1024
16
Cephalothin
4
128
1
Piperacillin
0.5
256
0.06
Cefotaxime
0.25
256
0.25
Ceftazidime
0.06
16
0.06
Aztreonam
0.25
0.25
0.06
Imipenem
0.06
0.12
0.015
Meropenem
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
0.25
0.25
0.25
Meropenem
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
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
Sensitive
cells killed
by 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
14/71
20
rd
No 3 gen ceph
Chow et al. Ann Intern Med 1991, 115, 585-90
Selecting Enterobacter R to
3rd gen cephs during Rx
Selection by cephs
Overall
Bacteraemia
Tissue, urine, wounds
Mortality
Mean stay
Median cost
Case
26%
29.5 days
$79,000
19%
29%
7%
Control
13%
19 days
$40,000
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
CIT
ENT
FOX
MOX
DHA
ACC
Source
C. freundii
Enterobacter spp.
Aeromonas spp.
Aeromonas spp.
M morganii
H. alvei
Examples
CMY-2 to 7; LAT-1,3,4
ACT-1; MIR-1
FOX-1 to -5;
MOX-1,-2; CMY-1,7 & 8
DHA-1, -2
ACC-1
Plasmid AmpC
Ampicillin
Co-amoxiclav
Cefotaxime
Ceftazidime
Ctaz + clav
Cefoxitin
Meropenem
b-Lactamases
MIC (mg/L)
K. pneum 77 E. coli R- E. coli R+
512
4
512
256
2
512
8
0.03
8
8
0.06
16
8
0.06
16
32
4
16
0.06
0.06
0.25
TEM-1, SHV-1
TEM-1,
AmpC
AmpC
Jenks et al. 1995. J Antimicrob Chemother 35, 235-6.
Enzymes in 1127 cephR isolates
from 16 labs in S England, 2004
600
CTX-M
500
Other ESBL
AmpC
Other
400
300
200
100
0
E. coli
K. pneumoniae
E. cloacae
Potz et al., JAC, 2006 in press
Prevalence of mechanisms: BSAC
bacteraemia surveillance, 2005
100%
80%
K1
Both
ESBL
AmpC
S
60%
40%
20%
0%
E. coli
Klebsiella
Enterobacter
http://www.bsacsurv.org
Inducible AmpC
Cefoxitin
But mutational
derepression is
the problem, not
induction
Ceftazidime
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 Enterobacter
AmpC
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 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
Cefotaxime combinations vs.
AmpC E. coli: London SE survey
30
Alone
+Clavulanate, 4 mg/L
+Cloxacillin 100 mg/L
25
20
15
10
5
MIC (mg/L)
4
>6
64
32
16
8
4
2
1
5
0.
25
0.
<=
0.
12
0
Cefotaxime combinations vs.
AmpC E. coli: London SE survey
70
Alone
+Clavulanate, 4 mg/L
+Cloxacillin 100 mg/L
60
50
40
30
20
10
MIC (mg/L)
4
>6
64
32
16
8
4
2
1
5
0.
25
0.
<=
0.
12
0
Cefotaxime / cloxacillin
tests for AmpC
MIC (mg/L)
1000
100
Cefotaxime
Cefotaxime +
cloxacillin
10
1
E. cloacae 684-con
P. aeruginosa 2297con
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 strain
E. cloacae,
AmpC
derepressed
Indicator
E. coli
NCTC10418
Disc
Cefoxitin 30 mg
Clover leaf (3 dimensional)
test for cephalosporinase
Test strain
E. cloacae,
AmpC
derepressed
Indicator
E. coli
NCTC10418
Disc
Cefotaxime 30 mg
Phenyl boronic acid for
detection of plasmid AmpC
Expansion (mm) of FOX
30 zone with 400 mg
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 % with >5 mm
reduced >4-fold by 100 mg/L cloxacillin zone expansion
Cefoxitin + cloxacillin 100 mg
86%
Cefoxitin + BZB 64 mg
89%
Cefpodoxime + BZB 64 mg
97%
Cefpodoxime + clav + BZB 64 mg
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, Phenyl boronic
disc
MIC
+
-
+
-
+
-
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
K. pneumoniae
Coudron JCM 2005 43 4163
Multiplex detection of
plasmid AmpC genes
Method of Perez-Perez & Hanson JCM 2002, 40, 2153
AmpC hyperproducers :
options
Active
Not active
Carbapenems
Penicillins except temocillin
Temocillin
Inhibitor combinations
4-gen cephs
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