AmpC b -Lactamases & their detection

1. AmpC b-Lactamases& their detection David Livermore Health Protection Agency, Colindale, London

2. H2N S N C N OR Oxyimino-aminothiazolyl stability to classical TEM/SHV b-Lactamase-stable cephs S CONH N O R COOH

3. b-Lactamase classes

4. 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

5. 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 AmpC -lactamases Derepressed Inducible Amt -lactamase [ -lactam]

6. 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

7. AmpD AmpR ampD ampR ampC Uninduced AmpC • Wall fragments recycled by AmpD • AmpRin repressor conformation • ampC (-lactamase gene) NOT expressed

8. Induced AmpC -lactamase AmpD ampD ampR ampC • More recycling: AmpD overwhelmed • Wall fragments convert AmpR to activator • ampC (-lactamase gene) expressed

9. Derepressed AmpC -lactamase++ ampD ampR ampC • ampD inactivated by mutation • AmpR constantly converted to activator • ampC hyper-expressed

10. Strong & weak inducers Strong inducer induces below MIC, weak doesn’t

11. MICs (mg/L) for E. cloacaeAmpC mutants

12. 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 MICs (mg/L) for P. aeruginosa AmpC mutants

13. 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

14. Over-run by mutants Mutant emerges randomly • 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... Sensitive cells killed by antibiotic Mutant’s progeny overrun

15. Initial isolation of 3rd-gen cephR Enterobacter, & prior Rx Chow et al. Ann Intern Med 1991, 115, 585-90

16. Selecting Enterobacter R to 3rd gen cephs during Rx Kaye et al. AAC 2001, 45, 2628; Cosgrove et al. Arch Intern Med 2002, 162, 185

17. Plasmidic AmpC enzymes • Escaping from enterobacterial and Aeromonas chromosomes • Many good reports since 1991 • CMY, MOX, FOX, LAT, DHA, ACT & BIL enzymes

18. Sources of plasmid AmpC

19. Plasmid AmpC Jenks et al. 1995. J Antimicrob Chemother35, 235-6.

20. Enzymes in 1127 cephR isolates from 16 labs in S England, 2004 Potz et al., JAC, 2006 in press

21. Prevalence of mechanisms: BSAC bacteraemia surveillance, 2005 http://www.bsacsurv.org

22. Inducible AmpC Cefoxitin But mutational derepression is the problem, not induction Better predict risk from species I/D Ceftazidime

23. Suspect derepressed / plasmid AmpC if: • Resistant 3-gen cephs, NOTcefepime & cefpirome • Resistant to cefoxitin (but more ESBL producers R, too, nowadays) • No ceph/clav synergy

24. Geometric mean MICs (mg/L): AmpC producers; 2004 London SE survey

25. 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

26. 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

27. Cefotaxime combinations vs. AmpC E. coli: London SE survey

28. Cefotaxime combinations vs. AmpC E. coli: London SE survey

29. Cefotaxime / cloxacillin tests for AmpC

30. 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

31. Clover leaf (3 dimensional) test for AmpC Test strain E. cloacae, AmpC derepressed Indicator E. coli NCTC10418 Disc Cefoxitin 30 mg

32. Clover leaf (3 dimensional) test for cephalosporinase Test strain E. cloacae, AmpC derepressed Indicator E. coli NCTC10418 Disc Cefotaxime 30 mg

33. Phenyl boronic acid for detection of plasmid AmpC Coudron JCM 2005 43 4163

34. Disc tests for AmpC BZB: benzo(b)thiophene-2-boronic acid Brenwaldet al., JAC 2005, 56, 600

35. Cefoxitin R isolates in phenyl boronic acid / cefoxitin tests Coudron JCM 2005 43 4163

36. Multiplex detection of plasmid AmpC genes Method of Perez-Perez & Hanson JCM 2002, 40, 2153

37. Active Carbapenems Temocillin 4-gen cephs Not active Penicillins except temocillin Inhibitor combinations 1, 2, 3-gen cephs Aztreonam AmpC hyperproducers : options

38. 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