Timothy R. Walsh, Cardiff University, Wales

(Plasmid mediated) Carbapenemases

What is a carbapenemase?

How much carbapenem do they need to breakdown beforethey are called a carbapenemase?

“ESBL-enzymes have relatively low hydrolytic capacitiesof carbapenems, we suggest that only enzymesdemonstrating imipenemase activity (Kcat/Vmax) ofgreater than 1 (μM-1 S-1)”

(Redefining extended-spectrum -lactamases: balancing science and clinical need.

Giske et al. JAC. 2009 63:1-4)

Are carbapenemases ESBLs? – moving onfrom 1989

GES-1, GES-2, GES-3, GES-4, GES-5 and GES-6 and GES-7 are ESBLs

GES-2, GES-4, GES-5 and GES-6 arecarbapenemases!!!!!!!!!!!!!!!!!!!!!!! AND GES-5 isreferred to in the literature as an ESBL

(Redefining extended-spectrum -lactamases: balancing science and clinical need.

Giske et al. JAC. 2009 63:1-4)

Carbapenemase Groups

• KPC (class A) – serine BL

• GES (class A) – serine BL

• OXA (class D) – serine BL

• Metallo--lactamases (class B)

blaKPC genes

• 10 derivatives (15/05/09)

• First identified in 1996 (USA)

• Found in USA, Puerto Rico, Israel, Colombia, Brazil,Argentina, China (Citrobacter freundii, E. coli andSerratia marcescens), Sweden, Norway, UKGreece/Turkey

• Associated with Tn3-type transposon (Tn4401)

(“The real threat of Klebsiella pneumonia carbapenemase producing bacteria”Nordmann, Cuzon & Nass. 2009. Lancet 9:228-36)

OXA-type carbapenemases

1. Walther-Rasmussen & Hoiby 2006. JAC. 57: 373-383.2. Poirel, L & Nordmann, P. 2006. CMI. 12:826-36.3. Poirel, L., Nass, T, Nordmann, P. 2009 (in press)

1

2

34

147 different variants (14/05/09)

Note: OXA24 and OXA40 are thesame. (www.lahey.org)

Table Susceptibility profile of metallo -β-lactamase–producing enterobacterial isolates .

MIC, μg/mL

Enterobacter cloacae

Method, antibiotic

Klebsiellapneumoniae,

KPMBL-A

(n= 14)

ECLMBL-1

(n = 2)

ECLMBL-2

(n = 2)

ECLMBL-3

(n= 2)

ECLMBL-4

(n = 1)

ECLMBL-5

(n = 1)

ECLMBL-6

(n = 3)

Klebsiellaoxytoca,

KOMBL-1

(n = 1)

Escherichiacoli,

ECMBL-1

(n = 1)

Microdilution

Piperacillin-tazobactam >64/4 >64/4 >64/4 >64/4 >64/4 >64/4 >64/4 >64/4 >64/4

Ceftazidime >16 >16 >16 >16 >16 >16 >16 >16 >16

Cefotaxime >8 >8 >8 >8 >8 >8 >8 >8 >8

Cefepime 8 to >16 16 to >16 >16 16 to >16 2 >16 16 to >16 >16 16

Imipenem 1 to >16 2–4 4 4 1 4 1 to 4 2 2

Meropenem 1 to 8 4 8 2–4 1 4 1 to 4 2 1

Tato et al., 2007. CID. 45: 1171-1178.

Do they always mediate resistance tocarbapenems?

Table Susceptibility profiles of the 17 gram-negative isolates carrying the blaVIM-1 gene.

MIC, μg/mL

Case Isolate IPM MEM ERT COL CIP GEN TIG

Antimicrobialresistance phenotypea ESBL presence

1 Enterobacter cloacae 1 1.5 4 0.38 0.38 0.5 0.5 ATM, ERT No

2 Klebsiella pneumoniae 1 2 3 1.5 8 0.5 0.25 ATM, TOB, TET, CIP Yes

3 K. pneumoniae 6 4 2 96 2 0.5 0.5 ATM, TOB, TET, CIP Yes

4 K. pneumoniae 3 3 2 0.19 12 0.5 0.25 ATM, TOB, CIP Yes

5 K. pneumoniae 2 6 3 128 >32 0.5 0.5 ATM, MEM, ERT, TOB, TET, CIP Yes

6 Enterobacter aerogenes 8 2 4 0.38 >32 2 0.5 TOB, CIP No

7 K. pneumoniae 12 3 3 ND >32 0.25 1 TOB, TET, CIP No

8 K. pneumoniae 8 8 8 0.5 >32 0.5 0.5 ATM, MEM, ERT, TOB, CIP Yes

9 K. pneumoniae 8 6 6 0.25 >32 0.5 2 ATM, TOB, TET, CIP Yes

10 K. pneumoniae 3 2 2 ND ND ND 0.5 ATM, ERT, TOB, CIP Yes

11 K. pneumoniae 2 4 6 0.5 >32 1 0.5 ATM, TOB, AMK, CIP Yes

12 K. pneumoniae 8 >32 16 0.5 >32 1 0.25 ATM, IPM, MEM, ERT, TOB, AMK, CIP Yes

13 Klebsiella oxytoca 1 1 0.5 0.25 16 8 1 TOB, TET, CIP No

14 K. pneumoniae 8 6 6 0.5 >32 0.5 0.5 ATM, IPM, MEM, ERT, TOB, TET, CIP Yes

15 K. pneumoniae >32 >32 >32 0.25 >32 8 1 ATM, IPM, MEM, ERT, TOB, TET, AMK, CIP Yes

16 K. pneumoniae 8 32 >32 0.5 >32 0.5 0.5 ATM, MEM, ERT, TOB, AMK, CIP Yes

17 K. pneumoniae 1.5 1.5 2 0.5 >32 1 1 ATM, ERT, TOB, TET, CIP Yes

Souli et al., 2008. CID. 46:847-854

In-vivo significance of MBLs mediating sub-carbapenem breakpoint MICs

832Kp-R

0.54Kp-1b

0.52Kp-1a

0.1250.125Kp-S

MerImiIsolate

Daikos et al., 2006. CMI.13: 202-205

Do they normally mediate resistance tocefotaxime and ceftazidime?

Table Susceptibility profile of metallo -β-lactamase–producing enterobacterial isolates .

MIC, μg/mL

Enterobacter cloacae

Method, antibiotic

Klebsiellapneumoniae,

KPMBL-A

(n= 14)

ECLMBL-1

(n = 2)

ECLMBL-2

(n = 2)

ECLMBL-3

(n= 2)

ECLMBL-4

(n = 1)

ECLMBL-5

(n = 1)

ECLMBL-6

(n = 3)

Klebsiellaoxytoca,

KOMBL-1

(n = 1)

Escherichiacoli,

ECMBL-1

(n = 1)

Microdilution

Piperacillin-tazobactam >64/4 >64/4 >64/4 >64/4 >64/4 >64/4 >64/4 >64/4 >64/4

Ceftazidime >16 >16 >16 >16 >16 >16 >16 >16 >16

Cefotaxime >8 >8 >8 >8 >8 >8 >8 >8 >8

Cefepime 8 to >16 16 to >16 >16 16 to >16 2 >16 16 to >16 >16 16

Imipenem 1 to >16 2–4 4 4 1 4 1 to 4 2 2

Meropenem 1 to 8 4 8 2–4 1 4 1 to 4 2 1

Tato et al., 2007. CID. 45: 1171-1178.

Do they always mediate resistance tocefotaxime and ceftazidime?

Where are they normally found?

++--NDM- MBL

--+++SPM- MBL

++/-++IMP- MBL

++/-++VIM- MBL

++++-OXA

+-+GES

+++--KPC

ColiformsACBPSA

blaKPC genes

• 10 derivatives (15/05/09)

• First identified in 1996 (USA)

• Found in USA, Puerto Rico, Israel, Colombia, Brazil,Argentina, China (Citrobacter freundii, E. coli andSerratia marcescens), Sweden, Norway, UKGreece/Turkey

• Associated with Tn3-type transposon (Tn4401)

(“The real threat of Klebsiella pneumonia carbapenemase producing bacteria”Nordmann, Cuzon & Nass. 2009. Lancet 9:228-36)

Species Distribution of Class D Carbapenemases

Cluster

Cluster 1 – plasmid mediated (Scaife et al., JAC. 1995. 36:585-586). Also,chromosome encoded in Proteus mirabilis (Bonnet et al., 2002. AAC.

46:2002-2004). Back to plasmid? (Niumsup et al., 2009. Jpn. J. infect Dis. 62:152-4)

Cluster 2 – First identified as chromosomal but now bothchromosomal and plasmid (Quinteira et al., 2007. AAC. 51:3465-3466). Foundrecently in Ps. aeruginosa on a plasmid (Poirel, personal communication,

Sevillano et al., 2008. Pathol Biol)

OXA-48 – Found in K. pneumoniae in Mediterranean basin onplasmids – IncN (Poirel et al., 2004. AAC. 48:15-22). Now spread to E. coliand Citrobacter spp.

OXA-58 – plasmid mediated and found only in Acinetobacter spp.(mainly A. baumannii)

Global Distribution of OXA-type Carbapenemases

Cluster 1 e.g. OXA-23

Cluster 2 e.g. OXA24-26, 40 etc.OXA-48

OXA-58

metallo--lactamases mimic ESBLs on manyissues not least detection in

Enterobacteriaceae

Pseudomonas aeruginosa

Acinetobacter spp. Enterobacteriaceae

? ?

Where are MBLs found?

Location of Mobile MBL- Containing Organisms

DIM-1 – Poirel and Nordmann (unpublished data)

TMB-1 – El Salabi, Toleman and Walsh (unpublished data)

NDM-1

IMP

VIMSPM-1

AIM-1GIM-1

SIM-1

TMB-1DIM-1

KHM-1

K. pneumoniae

E. coli

K. pneumoniae

E. coli

C. freundii

S. marcescens

Transfer of genesvia humanpopulationstypified byblaNDM-1

Are carbapenemase genes mainly foundon plasmids and is it important?

Conjugation

Plasmid mediated MBLs –what species are theyfound in?

• IMP – 1-24 (integron)• VIM- 1-22 (integron) (VIM-1 versus VIM-2 group!)• SPM-1• GIM-1 (integron)• SIM-1 (integron)• AIM-1• NDM-1 (integron)• KMH-1 (?) (Sekiguchi et al., AAC. 2008. 52:4194-4197)

• DIM-1 (integron) (Poirel & Nordmann – personal communication)

• TMB-1 (integron) (El Salabi et al. – unpublished)

Different means of transmission withinthe same city (Warsaw, Poland)

blaVIM-2 mostly found on plasmids

blaVIM-4 exclusively chromosomal

(Patzer and Toleman, unpublished data)

A)_

B)

Tn 501-like Replication/maintenance

Tn3-like Transposon Unknown

Transfer region

MerR MerT MerP MerA MerD MerE orf2 TnpR RepA ParC orf215 ParA1 orf217 ParA

TnpA IS aac blaVIM7 IntI TnpM

TrbL TrbK TrbJ TraI TraJ TraK KfrA

oriV

oriT

MerA

Orf-2

MerD

MerE

TnpR

RepA

ParC

Orf215

ParA1

Orf217

ParA

OrfL1Vim7IntI

p06-407.19TnpM

ParE

p07-406.21

p07-406.22

TrbL

TrbJ

TrbK

TraI

TraJ

TraKKfrA MerR MerT MerP

Pseudomonasplasmids containenvironmentalbacterial scaffoldscarryingresistance genes –success of VIM-2

IncP-like

Truncated traregionIintact Staregion

(Li et al., AAC. 2008. 52:3099-105)

K. pneumoniae

E. coli

K. pneumoniae

E. coli

C. freundii

S. marcescens

Transfer of genesvia humanpopulationstypified byblaNDM-1

Are they an indicator of an MDRphenotype?

• YES!

• Resistance profile usually includesaminoglycosides, all -lactams,fluoroquinolones

INT aacA7 blaVIM-2 aacC1 aacA4 qacEΔ1/sul

INT blaVIM-2 aacA4 qacEÄ1/sul

Genetic Elements in standard class 1 integrons

Resistance to:

Aminoglycosides

-lactams

Sulphamethoxazole

Disinfectants

(1-2kb)

Int1 arr-2 ereC aadA1 cmlA7 qacEΔ1 ISCR1

efflux pump Δldh ΔblaDHA-1ΔPAI blaNDM-1 ΔIS26 ΔTn3

A

B

ISEcP1 blaCMY-4 blc

(1-4kb)

Is there a limit to how much foreignDNA a bug can carry?

• We don’t know!!

Fournier et al. 2006. Comparative genomics of multi-drug resistance in Acinetobacter baumannii. Genetics. 2: 62-72

How complicated can it get?

We don’t know but mathematically it islikely to increase

Resistance/transfer occurs as a directresponse to antibiotic usage?

• Transfer? – quinolones?

• Spread of Resistance - probably

Is there a link between resistance andvirulence?

• We don’t know – BUT!

Lavigne et al., 2006. CMI

Carbapenemase Detection?

• Media, media, media!!!!

• DDS test

• Hodge Test

• Disk inhibitor test

• Inhibitor panels (false positive)

• Etest

General principal ofsubstrate and inhibitorcombinations.

For ESBLs:

Clavulanic acid

Sulbactum

Tazobactum

Enterobacter cloace IMP8

Klebsiella pneumoniae VIM-1

Klebsiella pneumoniae IMP-1

Escherichia coli ATCC 25922

What should we look out for?

S/I/RS/I/RRMeropenem

S/I/RS/I/RRImipenem

I/RRRErtapenem

RHigh RHigh RCTZ

RHigh RHigh RCTX

ColiformsACBPSA

What should we look out for?

• Battling against the complexities of BLs!

• Phoenix results

• CTX and CTZ resistance

• Ertapenem intermediate resistance

The only thing I am certain about is that Iknow nothing!

(Aristotle)

-And even then I’m not sure!

(Baquero 2008)