Detection of -lactamase-mediated resistance

David Livermore

Health Protection Agency,

Colindale, London

Main -lactamase threats

Extended-spectrum -lactamases

• TEM, SHV & CTX-M types

AmpC

• Derepressed chromosomal e.g Enterobacter

• Plasmid-mediated in E. coli & Klebsiella

Carbapenemases

• Metallo- & non-metallo-types

ESBL evolution

Activity vs3rd gen cephs

TEM-11964

TEM-21970

Gln39Lys

TEM-31987

Gln39Lys Glu104Lys Gly238Ser

MICs (mg/L) for ESBL- producing E. coli

R- TEM-1+ TEM-3+ TEM-10+

Ampicillin 2 1024 256 1024

Piperacillin 1 128 64 64

Pip + 4 mg/L taz 0.5 1 2 1

Ceftriaxone 0.03 0.03 64 2

Ceftazidime 0.12 0.12 32 128

Cefoxitin 4 4 8 4

Imipenem 0.12 0.12 0.12 0.12

Meropenem 0.03 0.03 0.03 0.03

Outcomes: infections with ‘ceph S’ ESBL producers

• Prospective study of K. pneumoniae bacteraemia & literature review

• 32 evalable patients with ceph ‘S/I’ ESBL producers

– 19/32 failed ceph Rx

Paterson et al. JCM 2001 39, 2206

• Bottom line- don’t use cephs vs. ESBL producers, even if they appear susceptible

Epidemiology of ESBL production

Pre –2000

• Mostly Klebsiella spp. with TEM/SHV

• Nosocomial, often ICU / specialist unit

• 1998: c. 25% of Klebs from European ICUs ESBL+

• 67% isolates outbreak strains; 33% non-outbreak

• Few epidemic strains

–- e.g K. pneumoniae K25 SHV-4+ in France

• Producers multi-R to quinolones & aminoglycosides

CTX-M -lactamases

• 37 types, 4 clusters

• Cefotaximases rather than ceftazidimases

• Predominant ESBLs in Argentina since 1990

– 75% of all ESBLs in Buenos Aires

• Disseminating rapidly now Asia & Europe

K. georgiana- related

K. ascorbata- related

CTX-M -lactamases

CTX-M in the UK

2000- First producers

K. oxytoca, Leeds, CTX-M-9

2001/2- First hospital outbreak

B’ham, 33 patients, K. pneumoniae, CTX-M-25

2001/2

CTX-M-15 in 4 / 922 E. coli from 3 / 28 hospitals

Brenwald JAC 2003, 51, 195; Alobwede JAC 2003, 51, 470: Mushtaq JAC 2003 52:528-9

2003 –repeated phone calls

‘We’ve got these ESBL producers from GP patients. About 20 or 30. Do you want them?’

“The patient hasn’t been in hospital…”

‘What do we use?- It’s got an ESBL & it’s trim and cipro resistant. We don’t want to have to admit the patient for i.v. therapy.’

“We don’t get bacteria like this from this sort of patient”

‘Will you I/D it? Our E. coli aren’t resistant like this.’ Is it an Enterobacter?’

UK, 2003-4: CTX-M-15 E. coli

• ARMRL rcvd >500 isolates form >75 UK labs

• Mix of hospital and community isolates

• Mostly urines; several bacteraemia admissions direct from community

• Most age >65; underlying problems, catheterised; hospital contact in past 0-3 years

Woodford et al. ECCMID, 2004

PFGE: CTX-M +ve E. coli

• 85% similarity = ‘strain’

• 65% isolates - 5 major strains

representatives all serotype O25

• epidemic strain A

110 isolates, 6 centres IS26 between blaCTX-M & normal

promotor

• 4 other major strains, B-E

• other isolates

Diverse/small clusters

Local epidemiology varies among centres

Lab RegionNo.

referred No.

strains% major

strains if >5%

1 W Mids 114 17 A, 61%; D, 18%

4 London 31 16 C 29%, E 9%

2 S East 26 1 A 100%

41 N Ireland 26 3 A 39%, C 50%

43 London 18 16 A, B both 5%

Geom. mean MICs, (mg/L) CTX-M-15 +ve E. coli

‘Epidemic A’ Other major Minor

Cefotaxime, 1 37.3 93.2 73.0

Ceftazidime, 2 2.9 23.0 37.9

Cefpodoxime, 1 49.7 233.9 256

Cephalexin, 32U 49.7 256 256

Co-amoxiclav, 16 18.1 20.1 17.0

Pip/taz, 16 20.1 13.2 14.7

Imipenem, 4 0.2 0.2 0.3

Ertapenem & meropenem also active

Geom. mean MICs, mg/L; UK CTX-M-15 producers

‘Epidemic’ A Other major Minor

Ciprofloxacin, 1 17.5 6.7 6.1

Trimethoprim, 2 256 9.6 45.3

Gentamicin, 1 1.1 28.6 12.2

Amikacin, 8 9.0 18.2 9.3

Fosfomycin, 128 0.9 0.6 1.9

Nitrofurantoin, 32 8 7.3 22.6

Spreading CTX-M

CTX-M-2: Israel

CTX-M-3: E. Europe, Far East

CTX-M-5: Latvia, salmonella

CTX-M-9/10-12 Spain

CTX-M-14: China

CTX-M-15: Canada, France, E. Europe (widely)

Russia- ‘CTX-M’s replacing TEM & SHV as the main ESBL types’

ECCMID 2004; ICAAC 2003; Rasmussen & Hoiby 2004 Can J Micro 50, 137.

17th July 2004: CTX-Mon Fleet St.

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

AmpC -lactamases

• Cephalosporins select derepressed mutants from inducible populations

• Selection c. 20% in Enterobacter bacteraemia

• 30-40% of all Enterobacter and C. freundii now derepressed at first isolation

• Resistant to inhibitors; escaping to plasmids

Acquired carbapenemases

IMP & VIM metallo--lactamases (Class B)

– Scattered reports- Far East; Europe

– Mostly in non-fermenters

Class A non-metallo--lactamases

– KPC small outbreaks in NE USA, Klebsiella & Enterobacter

– NMC/IMI in Enterobacter; SME in Serratia: v rare

Class D non-metallo--lactamases

– Important in Acinetobacter spp.

ESBL Detection: step 1

See http://www.hpa.org.uk

Screen Enterobacteriaceae with :

• Cefpodoxime- best general ESBL substrate

• Cefotaxime & ceftazidime- good substrates for CTX-M & TEM/SHV, respectively

Spread of CTX-M into community means screening must be wider than before

Detection of ESBLs: step 2

See http://www.hpa.org.uk

Seek ceph/clav synergy in ceph R isolates

•Double disc

•Combination disc

•Etest

ESBL detection : combination discs: +ve result, zone enlarged 50%

Discs (30+10 g) % Detected (n =100)

Ceftazidime +/- clav 88

Cefotaxime +/- clav 66

Both 93

M’Zali et al. 2000, JAC, 45, 881

0

10

20

30

40

50

60

-3 -1 1 3 5 7 9 11 13 15 17 19 21 23 25

Control AmpC K1 ESBL CTX-M

Zone differences (mm), Klebs & E. colic’pod/clav 10+1 g - c’pod 10 g

Etest for ESBLs

Cefotaxime

Cefotaxime+

clavulanate

Etest for ESBLs

Cefotaxime

Cefotaxime+

clavulanate

Pitfalls in ESBL detection

• Methods optimised for E. coli & Klebsiella

• More difficult with Enterobacter

– clavulanate induces AmpC; hides ESBL

• Do synergy test (NOT SCREEN) with 4th gen ceph

– but how sensitive are these for weak ESBLs?

Bacteria not to test for ESBLs

Acinetobacters

– Often S to clavulanate alone

S. maltophilia

– +ve result by inhibition of L-2 chromosomal -lactamase, ubiquitous in the species

Ceph R but synergy –ve…

AmpC- plasmid or chromosomal

S to 4 gen cephs; R to cefoxitin

K1 hyperproducer K. oxytoca

R cefuroxime, aztreonam, cefpodoxime

S ceftazidime, I to cefotaxime

May give false +ve ESBL test

Impermeable E. coli, Kleb

R cefoxitin & cefuroxime; not 3/4-gen cephs

Carbapenemase Metallo or not

R includes imipenem & / or meropenem

AmpC hyperproducing- how to confirm

• Resistant to 3rd gen cephs not cefepime

• No clavulanate synergy

• Cefoxitin R

• Enlarged zones to 3rd gen cephs if tests done on agar + 100 mg/L cloxacillin

• NOT just ‘because its an Enterobacter’

Double disc antagonismfor inducible AmpC

Cefoxitin Ceftazidime

AmpC inducibility- when to look

• Risk is mutation, not inducibility per se

• Best to identify & predict risk from species

• Just so ‘No’

• Warn clinicians against cephs for infections due to Enterobacter, C. freundii, Morganella & Serratia

Carbapenem resistance investigations

Enterobacteriaceae

Exceptional – needs ref. lab investigation

Acinetobacter spp.

Exceptional – needs ref lab investigation; PCR for Class D (OXA) -lactamase genes & MBL

P. aeruginosa

Low level (MIC <32 mg/L) – likely OprD loss

High level (MIC >32 mg/L) likely carbapenemase

Detecting class B enzymes:MBL Etests

• imipenem (I) vs. imipenem + EDTA (IPI)• ratio 8 consistent with MBL production• zone distortion consistent with MBL production• sensitivity - good ; specificity - poor

Why false +ves with Etest MBL?

EDTA may permeabilise the outer membrane

Zn++ suppresses OprD in P. aeruginosa, inducing imipenem resistance

–?? lack of zinc may induce OprD. Sensitising bug??

Zinc inactivates imipenem?2

1Carmen-Conjeho et al., ECCMID, 20032 Baxter & Lambert JAC 1997, 39, 838

MICs (mg/L) for E. cloacae with metallo--lactamases

MEM IMP AZT CTX CTZ CFM

R947 IMP-8TEM-1

1 2 0.03 >256 >256 32

Y580 IMP-8TEM-1

0.5 2 0.02 16 128 32

T524 IMP-8TEM-1

1 4 0.03 32 >256 32

N947C. freundii

VIM-2 0.5 1 0.06 32 64 16

Yan et al., JAC 2002, 50, 503

Some common questions 1

Can I use cephalexin in UTI screens, not cefpodoxime?

• No- some strain A CTX-M-15 +ve E. coli appear S

Can I project cefuroxime S/R from cefpodoxime?

• No: impermeable E. coli may be c’pod S; c’furox R

I use cefpirome/clav for confirmation with Enterobacter- can I use for all species?

• Not proven- not validated vs. weak producers

Some common questions 2

I can only have one plate per urine. What to test?

• C/pod, cipro, trim, nitro & 2 of amp, c/lex & Aug

How do I report cephs for ESBL producers?

• Resistant

How do I report -lactamase inhibitor combs?

• Arguable! Probably at face value….

Summary : -lactamase detection

Exploit indicator cephs

– Cefotaxime & ceftazidime OR cefpodoxime

– Cefepime/ cefpirome as stable to AmpC; cefoxitin to ESBL

– Use ceph / clav synergy tests to confirm ESBL producers

Avoid cephs vs. AmpC inducible Enterobacteriaceae

Use MBL Etests vs carbapenem R isolates,

– Be alert to false +ve results

Know patterns; spot the unusual & refer it!