CTXM ESBLs: molecular epidemiology of the current UK problem

1
CTX-M ESBLs molecular epidemiology of the
current UK problem

• Neil Woodford BSc PhD MRCPath
• Antibiotic Resistance Monitoring Reference
  Laboratory, SRMD - Colindale

2
CTX-M b-lactamases in the UK

• 2000 First producer (Alobwede JAC 2003, 51, 470)
• K. oxytoca, Leeds, CTX-M-09
• 2001-2 First hospital outbreak (Brenwald JAC
  2003, 51, 195)
• K. pneumoniae, Birmingham, CTX-M-25-like
• 2002 Surveillance isolates (Mushtaq JAC 2003,
  52, 528-9)
• 4 E. coli from 3 centres, CTX-M-15

3
Phylogeny of CTX-M ESBLs
4
ESBLs in UK E. coli 2003-4

• increasing reference requests for ESBL
  confirmation in E.coli
• most expressing phenotype consistent with CTX-M
  enzymes
• including isolates from community-acquired UTIs
• little recent hospital contact

5
17th July 2004 broadsheets discover CTX-M
6
ESBLs in UK E. coli 2003-4

• aim of this study was characterisation of
• isolates by PFGE (n 291 for this analysis)
• blaCTX-M alleles by PCR / sequencing

7
PFGE analysis of CTX-M-producing E. coli

• ?85 similarity to define strains
• 65 isolates belonged to 5 major strains
• representatives of all major strains serotype O25
• epidemic strain A
• 110 isolates, 6 centres
• specific molecular characteristics
• 4 other major strains
• other isolates
• many diverse strains
• some small (intra-hospital) clusters

8
Local epidemiology varies between centres
9
Characterisation of blaCTX-M alleles

• 291 isolates analysed
• 279 (96) contained alleles encoding group 1
  enzymes
• 12 contained alleles encoding group 9 enzymes
• 25 group 1 alleles sequenced
• 20 isolates from 17 centres blaCTX-M-15
  ISEcp1-like element
• 4 isolates from 3 centres blaCTX-M-15
  ISEcp1-like IS26 elements
• 1 isolate blaCTX-M-03 ISEcp1-like element

10
Disrupted blaCTX-M-15 in UK epidemic strain A
IS26 between the bla gene and its usual promoter

• lower MICs of CTX and CTZ likely due to altered
  expression
• PCR detection of this insertion
• working marker for this strain in the reference
  laboratory

11
Rapid PCR screening for probable strain A isolates

• amplify a 400-bp fragment
• spans the IS26-blaCTX-M link
• PFGE essential to confirm strain A
• other products possible
• care when interpreting
• only the 400-bp band is consistent with strain A
• distinct IS26-blaCTX-M links
• IS26-IS26 false products

12
3D clustering of selected CTX-M-15-producing
strains

• strains A, B, and D form distinct clusters
• 2 other strains have IS26 linked to blaCTX-M-15
• unrelated to strain A (TN17, from Paris, a
  minor UK strain)
• an IS26-blaCTX-M link is not a general indicator
  of strain relatedness

13
Geometric mean MICs, mg/L UK CTX-M producers
Meropenem ertapenem also active
14
Geometric mean MICs, mg/L UK CTX-M producers
15
Community-acquired CTX-M-producing strains

• 24 isolates, mainly from urines, referred as
  community-acquired or GP specimen
• 12 referring centres
• PFGE
• diverse types, but including
• strain A and all major strains
• CTX-M enzymes
• 67 produced CTX-M-15 / other group 1
• 3 produced CTX-M-09-like
• As multi-resistant as full collection

16
Producers of CTX-M-09-like enzymes

• 12 producers from 9 centres
• distinct strains, including
• 3 isolates from centre 4, 2 isolates from centre
  43
• unlinked emergence or horizontal transfer events ?

17
On-going work

• confirm whether UK blaCTX-M alleles are
  plasmid-mediated
• no transfer demonstrated to date (n 10
  isolates)
• evidence of horizontal transfer events ?
• further characterisation of strain A
• how does IS26 influence cephalosporin MICs ?
• does IS26 also affect gentamicin MIC, or is
  sensitivity an independent characteristic ?
• what factors contribute to the strains success ?
• how are O25 CTX-M-15-producing strains related ?
• investigate possible common ancestry

18
Reading List

• The UK situation
• Munday et al. JAC 2004 (Sept. issue)
• Woodford et al. JAC 2004 (Oct. issue)
• Reviews
• Bonnet. AAC 2004481-14
• Walther-Rasmussen Hoiby. Can J Microbiol
  200450137-165

19
Summary

• CTX-M-producing E. coli
• a rapidly developing problem (01/09/04 518
  producers from 77 labs)
• many produce CTX-M-15
• also detected in Klebsiella, Enterobacter,
  Citrobacter and Proteus (01/09/04 66 producers
  from 23 labs)
• complex epidemiology
• spread of major strains between centres
• local spread of major / minor strains
• multiple strains referred from some centres
• horizontal transfer of plasmids and / or
  integrons ?
• overspill into the community
• multi-resistance
• implications for treatment of community-acquired
  UTIs

20
Acknowledgements

• UK microbiologists
• vigilence
• referring isolates to ARMRL
• Control strains PCR conditions
• Guillaume Arlet
• Laurent Poirel
• Funding
• Health Protection Agency
• EU / FP6-funded COBRA project

• SRMD
• Elaina Ward, ARMRL
• Liz Fagan, ARMRL
• Edi Karisik, ARMRL
• Polly Kaufmann, LHCAI
• Jane Turton, LHCAI
• Tom Cheasty, LEP
• Marina Warner, ARMRL
• Rachel Pike, ARMRL
• Dot James, ARMRL