Detection of -Lactamase-Mediated Resistance

David M Livermore,Specialist & Reference

Microbiology Division

Action of a -lactamase

NO

COOH

S

HN

OCOOH

S

OH

Active penicillin

Inactive penicilloateH2O

-Lactamase families

Staph penicillinase; TEM & SHV; chromosomal -lactamases of Proteus, Klebsiella & Bacteroides

Zinc types

Chromosomal AmpC -lactamases of most enterobacteria

OXA-class plasmid -lactamases

AB

C

D

Established problems

•Staphylococcal penicillinase

•G-ve bacteria with penicillinases, TEM etc.

•G-ve bacteria with AmpC enzymes

•G-ve bacteria with TEM & SHV ESBLs

Other types emerging...

Emerging problems

•Plasmid AmpC enzymes

•ESBLs not derived from TEM & SHV

•Carbapenemases

•Resistance to inhibitor combinations

Spread of TEM plasmid -lactamases

• 1963 Ampicillin; 1st broad spectrum penicillin

• 1965 TEM -lactamases in E. coli

• 1969 TEM -lactamase in P. aeruginosa

• 1974 TEM in H. influenzae & N. gonorrhoeae

• Now TEM in 30-60% E. coli & enterobacteria & in 5-20% of H. influenzae & gonococci

-Lactamase stable cephs

CONHC

NOR

O

COOH

H2N

Oxyimino-aminothiazolylstability to classical TEM/SHV

OCH3

R

-methoxy, stability to TEM, SHV, ESBLs & Bacteroides enzymes

S NS

AmpC -lactamases

Basal in:• E. coli & shigellaeInducible in:• Enterobacter spp.• C. freundii• M. morganii• Serratia spp.• P. aeruginosa2nd, 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

Extended-spectrum -lactamases

TEM-11965

TEM-21970

TEM-31987

Activity vs.3rd gen cephs

and now up to TEM-126…, also SHV-48

MICs (mg/L) for ESBL +ve 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

Exotic ESBLs, not derived from TEM & SHV

• CTX-M- 29 variants, some derived from chromosomal -lactamase of K. ascorbata

– PER- PER-1 in Turkey; PER-2 in Argentina

– ESBL OXA-2 & -10 mutants- mostly P. aeruginosa Turkey

CTX-M -lactamases

• Escaped from the chromosomes of Kluyvera spp.

• More active vs. cefotaxime than ceftazidime

– But mutation can confer ceftazidimase activity

• Predominant ESBLs in Argentina since 1990

• Disseminating rapidly in Asia & Europe– 2003 ICAAC full of poster on ‘First CTX-M from ....’

Activity of CTX-M2Vmax

(%)Km

(M)MIC

R-

(mg/L)

MICR+

(mg/L)Cephaloridine 100 - - -

Piperacillin - - 1 1024

Cefotaxime 12.5 70 0.06 16

Ceftazidime 0.02 203 0.13 2

Imipenem 0 - 0.03 0.03

Bauernfeind et al., 1992 Infection 20, 158

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

• 2003- Community E. coli from UTIs

– Diverse strains & locales, 2 CTX-M variants

Brenwald JAC 2003, 51, 195; Alobwede JAC 2003, 51, 470: HPA data on file

Acquired carbapenemases rare

• BUT….

• IMP & VIM metallo-enzymes increasing,

• OXA carbapenemases in Acinetobacter spp.;

• Few class A types…. KPC, IMI, SME-

• Few major outbreaks of producers

VIM & IMP metallo--lactamases

• IMP, 16 types; VIM, 12 types; SPM-1

– 15% variation in families; 70% between them

• Hydrolyse -lactams except monobactams; inhibited by EDTA, not clav or sulphones

• Mostly Far East & S. Europe- few UK isolates

– Mostly P. aeruginosa; 2 Acinetobacter; 2 Klebsiella

• Not all gene +ve isolates are obviously R

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

-Lactamase detection

• Nitrocefin- very sensitive, expensive, good for fastidious GNB & Moraxella, not staph

• Acidimetric -sensitive, care with controls to avoid false +ve

• Iodometric -sensitive, fiddly, care with controls to avoid false +ve

• Microbiological -v. sensitive, slow

Challenges for the diagnostic lab

• Detection…. Haemophilus, Neisseria etc.

• Predicting -lactamase types. Have GNB got ?:

ESBL,

AmpC

Metallo types, VIM, IMP etc…

• Spotting unusual patterns; knowing what to refer

Some useful knowledge

AmpC

Hi-level

TEM ESBL

CTX-M K1

Ceftazidime R R v S

Cefotaxime R v R S

Cefoxitin R S S S

Aztreonam R v v R

Synergy + clav No +++ +++ No

Know the species

ESBLs: times a’ changing with CTX-M

• Old advice- test ceftazidime; ESBL test if R

• New advice- test ceftazidime & cefotaxime; ESBL test if R to either

• Alternative- test cefpodoxime; ESBL test if R

• Still true- Only testing cefuroxime is inadequate

ESBL detection

• Double disc synergy

• Combination discs

• E-test

– Test potentiation of ceftazidime, cefotaxime or cefpodoxime by clavulanate……

– Use whichever suggested ESBL production

Detection of ESBLs with combination discs (MAST)

+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), Klebsiella & E. coli

c’pod/clav 10+1 g minus c’pod 10 g

Etest for ESBLs

Cefotaxime

Cefotaxime+

clavulanate

Etest for ESBLs

Cefotaxime

Cefotaxime+

clavulanate

Difficulties in ESBL detection

• Ceph R Enterobacter etc. most likely AmpC derepressed ….. But may have ESBL

• Clav induces AmpC; hides ESBL inhibition

– Try to read anyway

– Suspect ESBL if pip/taz or cefotetan S

– Synergy test between cefepime & clav

Bacteria not to test for ESBLs

• Acinetobacters– Acinetobacters often S to clavulanate alone

• S. maltophilia– You get +ve results via inhibition of L-2

chromosomal -lactamase, which is ubiquitous in the species

AmpC inducibility- when to look

• Rarely!!!!!• Risk is mutation, not inducibility per se

• Best to identify & predict risk from species

• Biggest risk Enterobacter & C freundii

• Avoid cephalosporins against them

Identify means identify TO SPECIES LEVEL all Enterobacteriaceae (‘coliforms’) ex serious infections

AmpC & ESBLs- what to refer to ARMRL

• E. coli & Klebsiella suspected of AmpC

– ? Have plasmid types ? Recent travel

• ESBL +ves from community

– ? CTX-M types

• Enterobacters suspected of having ESBLs

Double disc antagonism for inducible AmpC

Cefoxitin Ceftazidime

Cheapskate’s insurance vs. lawyers for AmpC

derepression• Test cefoxitin

• Enterobacter & C. freundii with inducible AmpC are clearly R

• M. morganii & Serratia aren’t R; but carry lower derepression risk

• Species without inducible AmpC are S

ARMRL recommendations for carbapenem R isolates

• Enterobacteriaceae & Acinetobacter

– Send in to ARMRL

– Except Proteeae weakly R to imipenem only

• P. aeruginosa

– Screen with EDTA synergy test

– Send to ARMRL if +ve

S. maltophilia…. Please DON’T send

Etest for metallo--lactamase

Imipenem

Imipenem+

EDTA

Etest for metallo--lactamase

Imipenem

Imipenem+

EDTA

Carbapenem R isolates at ARMRL

• Screened with imipenem/EDTA Etest

• Spectrophotometry with imipenem

• PCR for carbapenemase genes

• DNA sequencing

Weaknesses of strategy• False positives with Etest MBL tests

– 9/23 MBL Etest+ve P. aeruginosa hydrolysis -ve & negative for blaIMP & blaVIM

• Class D -lactamases v. weak activity

– Difficult to detect hydrolysis

• Sometimes you wouldn’t guess to look!

Why false +ves with Etest MBL?

• EDTA may permeabilise the outer membrane

• Zinc suppresses OprD in P. aeruginosa, inducing imipenem resistance1 So?? lack of zinc may induce OprD. Sensitising the bug??

• Zinc inactivates imipenem!2

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

Activity of pip/tazo vs. ESBL +ve klebsiellae; 1994 & 1997/8

0

10

20

30

40

50

0.5 1 2 4 8 16 32 64 128 256 512 1024

MIC (mg/L)

% o

f E

SBL

pro

duce

rs

1994 1997/8

The message

• Beta-lactamases are getting more complex

• Full I/D needs complex molecular methods

Much can be inferred from simple tests.

Needs I/D

Testing wide panels of antibiotics; synergy tests

Knowledge of what’s unusual