Detection of blactamase mediated resistance

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Detection of b-lactamase-mediated resistance

• David Livermore
• Health Protection Agency,
• Colindale, London

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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

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ESBL evolution
Activity vs 3rd gen cephs
TEM-1 1964
Gln39?Lys
TEM-2 1970
Gln39?Lys
Glu104?Lys
Gly238?Ser
TEM-3 1987
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MICs (mg/L) for ESBL- producing E. coli
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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- dont use cephs vs. ESBL producers,
  even if they appear susceptible

Paterson et al. JCM 2001 39, 2206
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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

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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

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CTX-M b-lactamases
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CTX-M in the UK

• 2000- First producers
• K. oxytoca, Leeds, CTX-M-9
• 2001/2- First hospital outbreak
• Bham, 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 52528-9
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2003 repeated phone calls

• Weve got these ESBL producers from GP patients.
  About 20 or 30. Do you want them?

The patient hasnt been in hospital
Will you I/D it? Our E. coli arent resistant
like this. Is it an Enterobacter?
We dont get bacteria like this from this sort
of patient
What do we use?- Its got an ESBL its trim
and cipro resistant. We dont want to have to
admit the patient for i.v. therapy.
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UK, 2003-4 CTX-M-15 E. coli

• ARMRL rcvd gt500 isolates form gt75 UK labs
• Mix of hospital and community isolates
• Mostly urines several bacteraemia admissions
  direct from community
• Most age gt65 underlying problems, catheterised
  hospital contact in past 0-3 years

Woodford et al. ECCMID, 2004
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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

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Local epidemiology varies among centres
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Geom. mean MICs, (mg/L) CTX-M-15 ve E. coli
Ertapenem meropenem also active
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Geom. mean MICs, mg/L UK CTX-M-15 producers
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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-Ms replacing TEM SHV as the main
  ESBL types

ECCMID 2004 ICAAC 2003 Rasmussen Hoiby 2004
Can J Micro 50, 137.
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17th July 2004 CTX-M on Fleet St.
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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

Derepressed
Inducible
Amt ?-lactamase
? -lactam
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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

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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.

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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
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Detection of ESBLs step 2

• Seek ceph/clav synergy in ceph R isolates
• Double disc
• Combination disc
• Etest

See http//www.hpa.org.uk
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ESBL detection combination discs ve result,
zone enlarged 50
MZali et al. 2000, JAC, 45, 881
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Zone differences (mm), Klebs E. colicpod/clav
101 mg - cpod 10 mg
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Etest for ESBLs
Cefotaxime
Cefotaxime clavulanate
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Etest for ESBLs
Cefotaxime
Cefotaxime clavulanate
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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?

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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

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Ceph R but synergy ve
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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

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Double disc antagonism for inducible AmpC
Cefoxitin
Ceftazidime
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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

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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 lt32 mg/L) likely OprD loss
• High level (MIC gt32 mg/L) likely carbapenemase

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Detecting class B enzymesMBL Etests

• imipenem (I) vs. imipenem EDTA (IPI)
• ratio ?8 consistent with MBL production
• zone distortion consistent with MBL production
• sensitivity - good specificity - poor

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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
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MICs (mg/L) for E. cloacae with
metallo-b-lactamases
Yan et al., JAC 2002, 50, 503
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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 cpod S cfurox
  R
• I use cefpirome/clav for confirmation with
  Enterobacter- can I use for all species?
• Not proven- not validated vs. weak producers

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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.

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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!