Transcript Title of presentation

Detection of b-lactamasemediated 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
Carbapenemases
• Metallo- & non-metallo-types
ESBL evolution
Activity vs
3rd gen cephs
TEM-1
1964
Gln39Lys
TEM-2
1970
TEM-3
1987
Gln39Lys Glu104Lys
Gly238Ser
MICs (mg/L) for ESBLproducing E. coli
R-
TEM-1+ TEM-3+ TEM-10+
Ampicillin
2
1024
256
1024
Piperacillin
1
128
64
64
1
2
1
Pip + 4 mg/L taz 0.5
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
• Bottom line- don’t use cephs vs. ESBL
producers, even if they appear susceptible
Paterson et al. JCM 2001 39, 2206
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 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
CTX-M b-lactamases
K. georgiana- related
K. ascorbata- related
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…”
“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?’
‘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.’
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
No.
No.
% major
referred strains strains if >5%
114
17
A, 61%; D, 18%
Lab
Region
1
W Mids
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-M
on Fleet St.
AmpC b-lactamases
Basal in:
Derepressed
Inducible in:
Enterobacter spp.
C. freundii
M. morganii
Serratia spp.
P. aeruginosa
2nd, 3rd gen cephs:
Labile, but weak inducers, select
derepressed mutants
Amt b-lactamase
E. coli & shigellae
Inducible
[b -lactam]
AmpC b-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-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.
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
Detection of ESBLs: step 2
Seek ceph/clav synergy in ceph R isolates
• Double disc
• Combination disc
• Etest
See http://www.hpa.org.uk
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
Zone differences (mm), Klebs & E. coli
c’pod/clav 10+1 g - c’pod 10 g
60
Control
AmpC
K1
ESBL
CTX-M
50
40
30
20
10
25
23
21
19
17
15
13
11
9
7
5
3
1
-1
-3
0
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
b-lactamase, ubiquitous in the species
Ceph R but synergy –ve…
AmpC- plasmid or S to 4 gen cephs; R to cefoxitin
chromosomal
K1 hyperproducer R cefuroxime, aztreonam, cefpodoxime
K. oxytoca
S ceftazidime, I to cefotaxime
Impermeable
E. coli, Kleb
Carbapenemase
Metallo or not
May give false +ve ESBL test
R cefoxitin & cefuroxime; not 3/4-gen
cephs
R includes imipenem & / or meropenem
AmpC hyperproducinghow 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 antagonism
for 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) b-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, 2003
2 Baxter & Lambert JAC 1997, 39, 838
MICs (mg/L) for E. cloacae
with metallo-b-lactamases
MEM IMP AZT CTX CTZ CFM
R947
Y580
T524
N947
C. freundii
IMP-8
TEM-1
IMP-8
TEM-1
IMP-8
TEM-1
VIM-2
1
2
0.03 >256 >256
32
0.5
2
0.02
16
128
32
1
4
0.03
32
>256
32
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 b-lactamase inhibitor combs?
• Arguable! Probably at face value….
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!