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

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

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

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

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

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]

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 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

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

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

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 ....’

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

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

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-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

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

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

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

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

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% M’Zali et al. 2000, JAC, 45, 881

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

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 b-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-b-lactamase Imipenem Imipenem + EDTA

Etest for metallo-b-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 b-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, 2003 2 Baxter & Lambert JAC 1997, 39, 838

Activity of pip/tazo vs. ESBL +ve klebsiellae; 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