Detection of  -lactamase- mediated resistance David Livermore Health Protection Agency, Colindale, London

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

ESBL evolution Activity vs 3 rd gen cephs TEM TEM Gln39  Lys TEM Gln39  LysGlu104  LysGly238  Ser

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

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 R x Paterson et al. JCM , 2206 Bottom line- don’t use cephs vs. ESBL producers, even if they appear susceptible

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

CTX-M  -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

K. georgiana- related K. ascorbata- related CTX-M  -lactamases

CTX-M in the UK First producers K. oxytoca, Leeds, CTX-M /2- First hospital outbreak B’ham, 33 patients, K. pneumoniae, CTX-M /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 :528-9

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

UK, : 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

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 bla CTX-M & normal promotor 4 other major strains, B-E other isolates ­Diverse/small clusters

Local epidemiology varies among centres LabRegion No. referred No. strains % major strains if >5% 1W Mids11417A, 61%; D, 18% 4London3116C 29%, E 9% 2S East261A 100% 41N Ireland263A 39%, C 50% 43London1816A, B both 5%

Geom. mean MICs, (mg/L) CTX-M-15 +ve E. coli ‘Epidemic A’Other majorMinor Cefotaxime, Ceftazidime, Cefpodoxime, Cephalexin, 32U Co-amoxiclav, Pip/taz, Imipenem, Ertapenem & meropenem also active

Geom. mean MICs, mg/L; UK CTX-M-15 producers ‘Epidemic’ AOther majorMinor Ciprofloxacin, Trimethoprim, Gentamicin, Amikacin, Fosfomycin, Nitrofurantoin,

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 Micro 50, 137.

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

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

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

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

ESBL Detection: step 1 See 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

Detection of ESBLs: step 2 See Seek ceph/clav synergy in ceph R isolates Double disc Combination disc Etest

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

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

Etest for ESBLs Cefotaxime + clavulanate

Etest for ESBLs Cefotaxime + clavulanate

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 4 th gen ceph –but how sensitive are these for weak ESBLs?

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

Ceph R but synergy –ve… AmpC- plasmid or chromosomal S to 4 gen cephs; R to cefoxitin K1 hyperproducer K. oxytoca R cefuroxime, aztreonam, cefpodoxime S ceftazidime, I to cefotaxime May give false +ve ESBL test Impermeable E. coli, Kleb R cefoxitin & cefuroxime; not 3/4-gen cephs Carbapenemase Metallo or not R includes imipenem & / or meropenem

AmpC hyperproducing- how to confirm Resistant to 3 rd gen cephs not cefepime No clavulanate synergy Cefoxitin R Enlarged zones to 3 rd gen cephs if tests done on agar mg/L cloxacillin NOT just ‘because its an Enterobacter’

Double disc antagonism for inducible AmpC CefoxitinCeftazidime

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

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

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

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 1 Carmen-Conjeho et al., ECCMID, Baxter & Lambert JAC 1997, 39, 838

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

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

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  -lactamase inhibitor combs? Arguable! Probably at face value….

Summary :  -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!