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

Multiplex detection of plasmid AmpC genes

Method of Perez-Perez & Hanson JCM 2002, 40, 2153

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