1. Detection of b-Lactamase-Mediated Resistance
David M Livermore,
Specialist & Reference
Microbiology Division

2. Action of a b-lactamase
H2O N
Inactive penicilloate O
COOH S
Active penicillin HN O OH
COOH

3. A -Lactamase families C
Staph penicillinase; TEM & SHV; chromosomal -lactamases of Proteus, Klebsiella & Bacteroides
Zinc types
Chromosomal AmpC -lactamases of most enterobacteria
OXA-class plasmid -lactamases B C D

4. 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...

5. Emerging problems Plasmid AmpC enzymes
ESBLs not derived from TEM & SHV
Carbapenemases
Resistance to inhibitor combinations

6. 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

7. b-Lactamase stable cephs
a-methoxy, stability to TEM, SHV,
ESBLs & Bacteroides enzymes
H2N
OCH3 S N S C
CONH N OR O R
Oxyimino-aminothiazolyl
stability to classical TEM/SHV
COOH

8. AmpC -lactamases Basal in: Derepressed 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
Derepressed
Inducible
Amt -lactamase
[ -lactam]

9. 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

10. Extended-spectrum b-lactamases
Activity vs.
3rd gen cephs
TEM-1
1965
TEM-2
1970
TEM-3
1987
and now up to TEM-126…, also SHV-48

11. MICs (mg/L) for ESBL +ve E. coli

12. Exotic ESBLs, not derived from TEM & SHV
CTX-M- 29 variants, some derived from chromosomal b-lactamase of K. ascorbata
PER- PER-1 in Turkey; PER-2 in Argentina
ESBL OXA-2 & -10 mutants- mostly P. aeruginosa Turkey

13. CTX-M b-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 ....’

14. Activity of CTX-M2 V (%) K ( M) MIC R (mg/L) Cephaloridine 100
max
(%) K m ( M)
MIC R -
(mg/L) +
Cephaloridine
100
Piperacillin 1
1024
Cefotaxime
12.5 70
0.06 16
Ceftazidime
0.02
203
0.13 2
Imipenem -
0.03
0.03
Bauernfeind et al., 1992 Infection 20, 158

15. CTX-M in the UK 2000- First producers 2001/2- First hospital outbreak
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

16. 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

17. VIM & IMP metallo-b-lactamases
IMP, 16 types; VIM, 12 types; SPM-1
15% variation in families; 70% between them
Hydrolyse b-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

18. MICs (mg/L) for E. cloacae with metallo-b-lactamases
Yan et al., JAC 2002, 50, 503

19. b-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

20. Challenges for the diagnostic lab
Detection…. Haemophilus, Neisseria etc.
Predicting b-lactamase types. Have GNB got ?:
ESBL,
AmpC
Metallo types, VIM, IMP etc…
Spotting unusual patterns; knowing what to refer

21. Some useful knowledge Know the species AmpC Hi-level TEM ESBL CTX-M K1
Ceftazidime R v S
Cefotaxime
Cefoxitin
Aztreonam
Synergy + clav No
+++
Know the species

22. 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

23. ESBL detection Double disc synergy Combination discs E-test
Test potentiation of ceftazidime, cefotaxime or cefpodoxime by clavulanate……
Use whichever suggested ESBL production

26. Detection of ESBLs with combination discs (MAST) +ve result, zone enlarged 50%
M’Zali et al , JAC, 45, 881

27. Zone differences (mm), Klebsiella & E
Zone differences (mm), Klebsiella & E. coli c’pod/clav 10+1 mg minus c’pod 10 mg

28. Etest for ESBLs
Cefotaxime
Cefotaxime +
clavulanate

29. Etest for ESBLs
Cefotaxime
Cefotaxime +
clavulanate

30. 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

31. 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 b-lactamase, which is ubiquitous in the species

32. 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

33. 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

34. Double disc antagonism for inducible AmpC
Cefoxitin
Ceftazidime

35. 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

36. 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

37. Etest for metallo-b-lactamase
Imipenem
Imipenem +
EDTA

38. Etest for metallo-b-lactamase
Imipenem
Imipenem +
EDTA

39. Carbapenem R isolates at ARMRL
Screened with imipenem/EDTA Etest
Spectrophotometry with imipenem
PCR for carbapenemase genes
DNA sequencing

40. Weaknesses of strategy
False positives with Etest MBL tests
9/23 MBL Etest+ve P. aeruginosa hydrolysis -ve & negative for blaIMP & blaVIM
Class D b-lactamases v. weak activity
Difficult to detect hydrolysis
Sometimes you wouldn’t guess to look!

41. 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, 2003
2 Baxter & Lambert JAC 1997, 39, 838

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

43. 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