Activities of the National Reference Laboratory in UK Daniele - - PowerPoint PPT Presentation

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Activities of the National Reference Laboratory in UK Daniele Meunier HPA - ARMRL - Colindale Antibiotic Resistance Monitoring and Reference Laboratory - ARMRL National Reference Laboratory for the detection and investigation of

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Activities of the National Reference Laboratory in UK

Daniele Meunier

HPA - ARMRL - Colindale

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Antibiotic Resistance Monitoring and Reference Laboratory - ARMRL

  • National

Reference Laboratory for the detection and investigation

  • f

antibiotic resistance

  • f

public health importance

Antibiotic resistance and Evaluation Unit = AREU Resistance Mechanism Monitoring Unit = RMMU MICs of relevant antibiotics Investigation of resistance molecular mechanism

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Workflow

AREU / MICs Governance meeting RMMU / molecular mechanism Report to hospitals

  • MIC tests on isolates (agar broth dilution, E-tests
  • Confirmation of unusual resistances
  • Therapeutic guidance
  • Detection of carbapenemases production in

Pseudomonas spp, Acinetobacter spp and Enterobacteriaceae

  • Detection of cephalosporinases in Enterobacteriaceae
  • Detection of mecA and mupA in Staphylococci by PCR

and real-time PCR

  • Detection of a G2576T linezolid resistance mutation in

23S rRNA genes of Enterococci or Staphylococci

~ 100 isolates/week

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Determination of MICs

  • MICs by agar dilution
  • Interpretation

against BSAC and EUCAST guidelines, inferring resistance mechanisms by interpretative reading

  • MICs of a wide selection of relevant antibiotics for resistant isolates of

any species within our remit

  • MICs on antibiotics more suitable to detect resistance mechanisms

(carbenicillin in Pseudomonas…)

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Current methods for the detection

  • f antibiotic resistance genes
  • Simplex PCR for Ambler class A (KPC, SME, IMI, AIM, KHM, GES)

and class D (OXA-48) carbapenemases, class B metallo-β-lactamase (NDM)

  • Multiplex PCR for class B metallo-β-lactamase (GIM, IMP, VIM, SIM,

SPM)

  • Multiplex PCR for plasmidic AmpCs (ACC, CIT, DHA, EBC, FOX,

MOX)

  • Real-time PCR for detection of MecA/MupA
  • PCR-RFLP for linezolid
  • Time consuming, turnaround times to be reduced
  • Molecular tests done on ~ 30 isolates/wk, several tests (PCR) per

isolate to confirm or refute presence of carbapenemases

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Carbapenemase-producing Enterobacteriaceae in the UK

(n = 1003)

ARMRL, Unpublished data

Non-fermenters = 312 Enterobacteriaceae = 1003

  • KPC, OXA-48, NDM, VIM and IMP : top 5 carbapenemases in UK
  • Detection is diificult when co-resident mechanisms
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Need to improve our phenotypic detection methods

MIC (mg/L) All species 32 64 128 OXA-48 +ve 4 23 OXA-48 -ve 24 13 23

Temocillin vs OXA-48

MIC (mg/L) Species 8 16 32 64 128 Klebsiella spp

  • 31

27 15 2

  • E. coli

1 8 6

  • Enterobacter spp.

1

  • 2

1

  • Temocillin vs KPC

Livermore et al. IJAA, 37 (5): 415-9

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Need to improve our molecular detection methods

  • Evaluation of DNA microarray Check-points MDR CT102 : detection of

KPC, OXA-48, NDM-1, GIM, IMP and ESBLs (CTX-M group, SHV, TEM) One array per 3 samples In validation process

  • Maldi-TOF : carbapenemase detection
  • Luminex assay for detection of carbapenemases :

multiplex PCR of the regions of interest + primer extension step specific of the allele

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KPC +ve bacteria in the UK (Jan ‘12)

  • 621 x Enterobacteriaceae
  • 520 x Klebsiella spp.
  • 50 x E. coli
  • 40 x Enterobacter spp.
  • 9 x Raoutella spp.
  • 1 x Citrobacter spp.
  • 1 x Serratia sp.

Scattered, but dominant in NW England ...c. 75% of all isolates

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International strain epidemic: KPC +ve K. pneumoniae

ST258 ST258 ST258 Nordmann et al. TLID 2009; 9: 228–36

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..., but in NW England it’s different

ST258 ST258 ST258 One KPC outbreak: 11, 25, 27. 248, (258 - Col-R)

  • 321. 468. 490, 491

plus Enterobacter + E. coli Nordmann et al. TLID 2009; 9: 228–36

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KPC outbreak - NW England: hypertransmissible IncFII plasmid

  • E. coli

Enterobacter ST258, hosp 2 ST258, hosp 1 ST25, hosp 1 ST468, hosp 1 ST11, hosp 1

  • K. pneumoniae

Self-replicating sub-unit Sequence divergence In NW

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OXA-48-like enzymes in the UK (Jan ‘12)

  • 92 x Enterobacteriaceae
  • 72 x Klebsiella spp.
  • 17 x E. coli
  • 2 x Enterobacter spp.
  • 1 x Citrobacter freundii

Spread of highly related plasmids

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Outbreak strain (ST353)

OXA-48-producing K. pneumoniae in the UK

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International plasmid ‘epidemic’: OXA-48 plasmids in Klebsiella, Enterobacter and E. coli

OXA-48 (c. 62 kb) OXA-48 OXA-181 (c. 7kb)

Dimou et al. JAC 2012 ; Poirel et al. AAC 2012

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NDM +ve bacteria in the UK (115 patients, Jan ‘12)

  • 138 x Enterobacteriaceae
  • 87 x Klebsiella spp.
  • 33 x E. coli
  • 12 x Enterobacter spp.
  • 3 x Citrobacter spp.
  • 1 x Morganella morgannii
  • 1 x Providencia sp.
  • 1 x Serratia sp.
  • 11 x A. baumannii

NDM-1 is coded by a mobile gene, on mobile plasmids, and will spread further

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blaNDM is on diverse plasmids

  • IncA/C >> IncL/M, IncN, IncFI/II or NT
  • Variation can be introduced during transfer / cloning

Kumarasamy et al., Lancet ID 2010; 10: 597 Poirel et al. AAC 2010; 54: 4914

1 2

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ARMRL in national and European surveillance

  • ARMRL undertakes sentinel surveys of resistance
  • BSAC surveillance is sub-contracted to ARMRL
  • ARMRL is partner in GRASP (Gonococcal Resistance to

Antibiotics Surveillance Programme)

  • ARMRL

works in close relationship with Salmonella Reference Unit in HPA – Colindale

  • ARMRL is major partner of EARSS
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Bacteraemia as a mandatory national surveillance

  • 2173 E. coli bacteraemia isolates from 2001-2010 from 18

centres across UK and Ireland

  • Phylotyping based on the Clermont et al. typing scheme

(B2 most common group found)

  • MLST using the Achtman scheme
  • PFGE, virulence array, whole genome sequencing will be

done on a subset of isolates

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Which E. coli clones cause bacteraemia (and UTI)?

  • six major types increased from 45.6% to 64.4%, 2001 - 2010
  • ST131 - most significant increase
  • ST73 and ST95 not in ESBL +ve strain sets ...why ?

ST Bacteraemia 2001 (n=152) Bacteraemia 2010 (n=116) UTIs (n=300)* 73 20.5% 21.2% 16.6% 95 7.9% 11.0% 6.3% 131 3.3% 13.6% 12.3% 12 5.3% 7.6% 0.7% 127 4.6% 5.9% 3.6% 69 4.0% 5.1% 9.0% Other 54.4% (58 STs) 35.6% (30 STs) 51.5% (97 STs)

* Gibreel et al. JAC 2012; 67: 346-56

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ARMRL – Research and development

  • Investigation of resistance to 'antibiotics of last resort' i.e. to drugs that usually

remain active against bacteria resistant to other agents. For gram-positive bacteria: oxazolidinones (linezolid), lipopeptides (daptomycin) and glycylcycline (tigecycline). For gram-negative bacteria: imipenem, meropenem, and ertapenem, and the glycylcycline, tigecycline.

  • ARMRL's research interests are focused on :

mechanisms of non-carbapenemase-mediated resistance to carbapenems in Enterobacteriaceae, particularly in Klebsiella spp. and Enterobacter spp. mechanisms of resistance or reduced susceptibility to tigecycline in Enterobacteriaceae and Acinetobacter spp.

  • the application of proteomics techniques to investigate novel and complex

resistance phenotypes.

  • Evaluation of new antibacterials in collaboration with the pharmaceutical

companies.

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Thank you…

Antibiotic Resistance Monitoring & Reference Laboratory 61 Colindale Avenue London NW9 5EQ Tel +44 (0)20 8327 6511 www.hpa.org.uk email: armrl@hpa.org.uk