detection of b -lactamase- mediated resistance
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
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 vs3rd gen cephs
TEM-11964
TEM-21970
Gln39Lys
TEM-31987
Gln39Lys Glu104Lys Gly238Ser
MICs (mg/L) for ESBL- producing E. coli
R- TEM-1+ TEM-3+ TEM-10+
Ampicillin 2 1024 256 1024
Piperacillin 1 128 64 64
Pip + 4 mg/L taz 0.5 1 2 1
Ceftriaxone 0.03 0.03 64 2
Ceftazidime 0.12 0.12 32 128
Cefoxitin 4 4 8 4
Imipenem 0.12 0.12 0.12 0.12
Meropenem 0.03 0.03 0.03 0.03
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
Paterson et al. JCM 2001 39, 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
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
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, 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
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
Local epidemiology varies among centres
Lab RegionNo.
referred No.
strains% major
strains if >5%
1 W Mids 114 17 A, 61%; D, 18%
4 London 31 16 C 29%, E 9%
2 S East 26 1 A 100%
41 N Ireland 26 3 A 39%, C 50%
43 London 18 16 A, B both 5%
Geom. mean MICs, (mg/L) CTX-M-15 +ve E. coli
‘Epidemic A’ Other major Minor
Cefotaxime, 1 37.3 93.2 73.0
Ceftazidime, 2 2.9 23.0 37.9
Cefpodoxime, 1 49.7 233.9 256
Cephalexin, 32U 49.7 256 256
Co-amoxiclav, 16 18.1 20.1 17.0
Pip/taz, 16 20.1 13.2 14.7
Imipenem, 4 0.2 0.2 0.3
Ertapenem & meropenem also active
Geom. mean MICs, mg/L; UK CTX-M-15 producers
‘Epidemic’ A Other major Minor
Ciprofloxacin, 1 17.5 6.7 6.1
Trimethoprim, 2 256 9.6 45.3
Gentamicin, 1 1.1 28.6 12.2
Amikacin, 8 9.0 18.2 9.3
Fosfomycin, 128 0.9 0.6 1.9
Nitrofurantoin, 32 8 7.3 22.6
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.
17th July 2004: CTX-Mon 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]
Am
t -
lact
am
ase
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 http://www.hpa.org.uk
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 http://www.hpa.org.uk
Seek ceph/clav synergy in ceph R isolates
•Double disc
•Combination disc
•Etest
ESBL detection : combination discs: +ve result, zone enlarged 50%
Discs (30+10 g) % Detected (n =100)
Ceftazidime +/- clav 88
Cefotaxime +/- clav 66
Both 93
M’Zali et al. 2000, JAC, 45, 881
0
10
20
30
40
50
60
-3 -1 1 3 5 7 9 11 13 15 17 19 21 23 25
Control AmpC K1 ESBL CTX-M
Zone differences (mm), Klebs & E. colic’pod/clav 10+1 g - c’pod 10 g
Etest for ESBLs
Cefotaxime
Cefotaxime+
clavulanate
Etest for ESBLs
Cefotaxime
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 4th 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 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’
Double disc antagonismfor inducible AmpC
Cefoxitin Ceftazidime
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
1Carmen-Conjeho et al., ECCMID, 20032 Baxter & Lambert JAC 1997, 39, 838
MICs (mg/L) for E. cloacae with metallo--lactamases
MEM IMP AZT CTX CTZ CFM
R947 IMP-8TEM-1
1 2 0.03 >256 >256 32
Y580 IMP-8TEM-1
0.5 2 0.02 16 128 32
T524 IMP-8TEM-1
1 4 0.03 32 >256 32
N947C. freundii
VIM-2 0.5 1 0.06 32 64 16
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!