1 / 41

Detection of b -lactamase- mediated resistance

Detection of b -lactamase- mediated resistance. David Livermore Health Protection Agency, Colindale, London. Main b -lactamase threats. Extended-spectrum b -lactamases TEM, SHV & CTX-M types AmpC Derepressed chromosomal e.g Enterobacter Plasmid-mediated in E. coli & Klebsiella

eshe
Download Presentation

Detection of b -lactamase- mediated resistance

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Detection of b-lactamase-mediated resistance David Livermore Health Protection Agency, Colindale, London

  2. Main b-lactamase threats • Extended-spectrum b-lactamases • TEM, SHV & CTX-M types • AmpC • Derepressed chromosomal e.g Enterobacter • Plasmid-mediated in E. coli & Klebsiella • Carbapenemases • Metallo- & non-metallo-types

  3. ESBL evolution Activity vs 3rd gen cephs TEM-1 1964 Gln39Lys TEM-2 1970 Gln39Lys Glu104Lys Gly238Ser TEM-3 1987

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

  5. 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 • Bottom line- don’t use cephs vs. ESBL producers, even if they appear susceptible Paterson et al.JCM 2001 39, 2206

  6. 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

  7. CTX-M b-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

  8. K. georgiana- related K. ascorbata- related CTX-M b-lactamases

  9. 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

  10. 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…” ‘Will you I/D it? Our E. coli aren’t resistant like this.’ Is it an Enterobacter?’ “We don’t get bacteria like this from this sort of patient” ‘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.’

  11. 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

  12. 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

  13. Local epidemiology varies among centres

  14. Geom. mean MICs, (mg/L) CTX-M-15 +ve E. coli Ertapenem & meropenem also active

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

  16. 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 Micro50, 137.

  17. 17th July 2004: CTX-M on Fleet St.

  18. 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]

  19. 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

  20. Acquired carbapenemases • IMP & VIM metallo-b-lactamases (Class B) • Scattered reports- Far East; Europe • Mostly in non-fermenters • Class A non-metallo-b-lactamases • KPC small outbreaks in NE USA, Klebsiella & Enterobacter • NMC/IMI in Enterobacter; SME in Serratia: v rare • Class D non-metallo-b-lactamases • Important in Acinetobacter spp.

  21. ESBL Detection: step 1 • 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 See http://www.hpa.org.uk

  22. Detection of ESBLs: step 2 • Seek ceph/clav synergy in ceph R isolates • Double disc • Combination disc • Etest See http://www.hpa.org.uk

  23. ESBL detection : combination discs: +ve result, zone enlarged 50% M’Zali et al. 2000, JAC, 45, 881

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

  25. Etest for ESBLs Cefotaxime Cefotaxime + clavulanate

  26. Etest for ESBLs Cefotaxime Cefotaxime + clavulanate

  27. 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?

  28. Bacteria not to test for ESBLs • Acinetobacters • Often S to clavulanate alone • S. maltophilia • +ve result by inhibition of L-2 chromosomal b-lactamase, ubiquitous in the species

  29. Ceph R but synergy –ve…

  30. 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’

  31. Double disc antagonism for inducible AmpC Cefoxitin Ceftazidime

  32. 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

  33. Carbapenem resistance investigations • Enterobacteriaceae • Exceptional – needs ref. lab investigation • Acinetobacter spp. • Exceptional – needs ref lab investigation; PCR for Class D (OXA) b-lactamase genes & MBL • P. aeruginosa • Low level (MIC <32 mg/L) – likely OprD loss • High level (MIC >32 mg/L) likely carbapenemase

  34. 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

  35. 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, 2003 2 Baxter & Lambert JAC 1997, 39, 838

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

  37. 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

  38. 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 b-lactamase inhibitor combs? • Arguable! Probably at face value….

  39. Summary : b-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!

More Related