Presentation is loading. Please wait.

Presentation is loading. Please wait.

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

Similar presentations

Presentation on theme: "Detection of  -Lactamase- Mediated Resistance David M Livermore, Specialist & Reference Microbiology Division."— Presentation transcript:

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

2 Action of a  -lactamase N O COOH S HN O COOH S OH Active penicillin Inactive penicilloate H2OH2O

3  -Lactamase families Staph penicillinase; TEM & SHV; chromosomal  -lactamases of Proteus, Klebsiella & Bacteroides Zinc types Chromosomal AmpC  -lactamases of most enterobacteria OXA-class plasmid  -lactamases A 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 1974TEM in H. influenzae & N. gonorrhoeae NowTEM in 30-60% E. coli & enterobacteria & in 5-20% of H. influenzae & gonococci

7  -Lactamase stable cephs CONHC N OR O COOH H2NH2N Oxyimino-aminothiazolyl stability to classical TEM/SHV OCH 3 R  -methoxy, stability to TEM, SHV, ESBLs & Bacteroides enzymes SN S

8 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] Amt  -lactamase Derepressed Inducible

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  -lactamases TEM-1 1965 TEM-2 1970 TEM-3 1987 Activity vs. 3rd gen cephs 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  -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  -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 Ceftazidime0.022030.132 Imipenem0-0.03 Bauernfeind et al., 1992 Infection 20, 158

15 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

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-  - 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

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

19  -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  -lactamase types. Have GNB got ?:  ESBL,  AmpC  Metallo types, VIM, IMP etc … Spotting unusual patterns; knowing what to refer

21 Some useful knowledge AmpC Hi-level TEM ESBL CTX-MK1 CeftazidimeRRvS CefotaximeRvRS CefoxitinRSSS AztreonamRvvR Synergy + clavNo+++ 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. 2000, JAC, 45, 881

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

28 Etest for ESBLs Cefotaxime + clavulanate

29 Etest for ESBLs 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  -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-  -lactamase Imipenem + EDTA

38 Etest for metallo-  -lactamase 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 bla IMP & bla VIM Class D  -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 resistance 1 So?? lack of zinc may induce OprD. Sensitising the bug?? Zinc inactivates imipenem! 2 1 Carmen-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

Download ppt "Detection of  -Lactamase- Mediated Resistance David M Livermore, Specialist & Reference Microbiology Division."

Similar presentations

Ads by Google