Genetics structures at the origin of acquisition of the carbapenemase

genes

Laurent Poirel

South-Paris Medical School INSERM U914 « Emerging Resistance to Antibiotics »

Hospital Bicêtre France

ESCMID Course, Athens 2011

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Variety of carbapenemase genes

• Class A; KPC, GES-like

• Class B; IMP, VIM, NDM, GIM

• Class D; OXA-23, OXA-40, OXA-58 OXA-48, OXA-181

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Variety of carbapenemase genes

• Class A; KPC, GES-like mostly Enterobacteriaceae, but also Pseudomonas

• Class B; IMP, VIM, NDM Enterobacteriaceae, Pseudomonas, and Acinetobacter

• Class D; OXA-23, OXA-40, OXA-58 Acinetobacter

OXA-48, OXA-181 Enterobacteriaceae

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Variety of genetic elements involved in mobility of ß-lactamase genes

• Transposons Class II transposons (Tn3-type) Composite transposons Conjugative transposons

• Integrons Class 1 integrons

• Insertion sequences

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Tn3-type transposons

• 2 extremities corresponding to inverted repeat sequences (IR) • 1 transposase (TnpA) => recognizes IR extremities in the

transposition process and cuts target DNA • 1 resolvase (TnpR) : negative regulator of TnpA expression and

recombinase mediating the resolution of the transposon integration process

res tnpA blaTEM Tn1

IRL IRR

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Insertion sequences (IS)

IRL IRR

mARN

Transposase

ATGCA TACGT

ATGCA TACGT

ATGCA TACGT

Target site

IS

gene

IS

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The blaKPC-borne transposon

ATTGA ATTGA ISKpn7 ISKpn6

IstB IstA

tnpA tnpA tnpR

blaKPC-2

Tn4401

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CCG CCG TA TA

KPC-2 IstB IstA

tnpA tnpR

ATTGA ATTGA

KPC-2

tnpA tnpR

tnpA tnpR KPC-2

ATTGA

ISKpn7 ISKpn6

IRL IRR1

IRR

IRL

IRR1

IRR

IRL IRR

A

B

C ISKpn7 ISKpn6

Genesis of Tn4401

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GCGCT GCGCT ISKpn7 ISKpn6

IstB IstA

tnpA tnpA tnpR

blaKPC-2

ATTAC ATTAC ISKpn7 ISKpn6

IstB IstA

tnpA tnpA tnpR

blaKPC-2

TTGGT TTGGT ISKpn7 ISKpn6

IstB IstA

tnpA tnpA tnpR

blaKPC-2

ATTGA ATTGA ISKpn7 ISKpn6

IstB IstA

tnpA tnpA tnpR

blaKPC-2

ATTGA ATTGA ISKpn7 ISKpn6

IstB IstA

tnpA tnpA tnpR

blaKPC-2

P. aeruginosa from Colombia

K. pneumoniae from Colombia

K. pneumoniae from Colombia

K. pneumoniae from Greece

K. pneumoniae from NYC

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The class 1 integrons

intI1

attI

sul1 qacE∆

resistance gene cassette (R gene)

integrase

Int

3'- conserved segment

attC

ant P

ant P

attI intI1 sul1 qacE∆ R gene attC

R gene attC

R gene attC

• Consequences : co-resistance ; co-expression ; co-selection

5'- conserved segment VARIABLE

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Structure of a gene cassette

Gene TTAAGC-- GCTTAAC---//-- --G GTTRRRY--

RBS

ATG TAA

gene cassette

59-be

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Acquisition of resistance genes as a form of gene cassettes

ß-lactams => blaVEB, blaGES, blaOXA, blaPSE,… Aminoglycosides => many genes aac(6’), aadA… Chloramphenicol => cmlA Sulfonamides => sul Trimethoprim => dfr Rifampicin => arr

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Integrons are carried by transposons

intI1 qacE D 1 sul1 orf5

Tn21 tnpA tnpR tnp

M

res

urf2 E D A

C P T R

The integron is not self-mobile, it is mobilized by the transposon

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blaGES-like integron structures

blaGES-2

P2

int1 qacE∆1 sul1

qacE∆1 sul1 blaGES-2

P2

int1 blaOXA-5 aac(3)-I

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Integron-borne blaVIM-1

The carbapenemase gene is usually identified in association with aminoglycosides resistance genes, and eventually with additional ß-lacrtamse genes of narrow

spectrum

aacA4 blaVIM-1 blaOXA-2 sul1 intI1 aadA1

Similar features for the widespread blaIMP genes

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Insertion sequences (IS)

IRL IRR

mARN

Transposase

ATGCA TACGT

ATGCA TACGT

ATGCA TACGT

Target site

IS

gene

IS

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Genesis of composite transposons (I)

ATGCA TACGT

ATGCA TACGT

ATGCA TACGT

Target site

IS

IS

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ACGGA TGCCT

ATGCA TACGT

ATGCA TACGT

Target site IS

IS

Genesis of composite transposons (II)

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ATGCA TACGT

ATGCA TACGT IS IS

• Composite transposon = two IS bracketing the transposed fragment

Resistance gene

ACGGA TGCCT

ACGGA TGCCT

Genesis of composite transposons (III)

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ATGCA TACGT

ATGCA TACGT

IS IS

• Composite transposon = two IS bracketing the transposed fragment

Resistance gene

ACGGA TGCCT

ACGGA TGCCT

Genesis of composite transposons (IV)

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The blaOXA-48 gene is part of a composite transposon

• The blaOXA-48 gene, firstly in Klebsiella pneumoniae, is widespread in Turkey

• OXA-48 hydrolyzes carbapenems, but spares ceftazidime and cefotaxime • Responsible for carbapenem resistance in Klebsiella pneumoniae, Citrobacter freundii, and Escherichia coli

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The blaOXA-48 gene is part of a composite transposon

Prom

IS1 999 IS1 999 blaOXA-48

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Prom1

IS1 999 IS1 999 blaOXA-48

blaOXA-48 gene expression can be modulated

Prom2

∆IS1 999 IS1 999 blaOXA-48

AAGAATGTT AAGAATGTT

IS1 R

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Prom

IS1 999 IS1 999 blaOXA-48

Shewanella spp.

A Gram negative species isolated from lake sediments

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The blaOXA-23 gene is associated to ISAba1 elements

ISAba1 blaOXA-23

ATTCGCGGG ATTCGCGGG

sulf.

Tn2006

ISAba1 ATPase sulf.

Acquired resistance to carbapenems in A. baumannii

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ISAba1 blaOXA-23DEAD

ATTCGCGGG ATTCGCGGG

sulf.

Tn2006

ISAba1ATPase

DNA methyl

sulf.ISAba1 blaOXA-23DEAD

ATTCGCGGG ATTCGCGGG

sulf.

Tn2006

ISAba1ATPase

DNA methyl

sulf.

blaOXA-23 has been captured from the A. radioresistens chromosome

Acinetobacter radioresistens

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One single IS element may mobilize the resistance genes

ISEcp1

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ISEcp1

IRL IRR tnpA

ISEcp1

• Belongs to the IS1380 family

• Possesses two imperfect 18-bp inverted repeats

• One transposase of 420 amino acid long, sharing 41% with that of IS1380

• Generates 5-bp duplication of the target site upon transposition

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blaCTX-M IRR-like

IRL IRR tnpA

ISEcp1

ISEcp1 recognizes variable IRR sequences during its transposition process

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ISEcp1 and other ß-lactamase genes

Involved in the acquisition of plasmid-borne CTX-M and AmpC-type ß-lactamase genes blaCTX-M (originating from Kluyvera spp.) blaCMY (originating from Citrobacter freundii) blaDHA (originating from Morganella morganii) blaACC (originating from Hafnia alvei)

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Different mobilization events for closely-related blaCTX-M genes

tnpA

ISEcp1

IRL IRR blaCTX-M-15

tnpA

ISEcp1

IRL IRR blaCTX-M-3

48 bp

128 bp

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Role of ISEcp1 in expression of ß-lactamase genes

tnpA

ISEcp1

IRL IRR bla

-35 -10

A so-called « strong » promoter

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blaOXA-181 ISEcp1

CCTAGATTCTACGT ACATGAATCCTCCG

ATATA ATATA

Tn2013

ACGTGGAATTTAGG

IRL IRR1 IRR2

∆lysR

∆ere

Genetic environment of blaOXA-181

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Genetic environment of blaOXA-181 compared to that of blaOXA-48

blaOXA-181 ISEcp1

Tn2013

∆lysR

∆ere

blaOXA-48 IS1999

Tn1999

IS1999 lysR

Kp 11978

Kp 481814

94% identité

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Genetic environment of blaOXA-181

blaOXA-181 ISEcp1

Tn2013

∆lysR

∆ere Kp 481814

The transposition of Tn2013 was demonstrated in-vitro

Potron et al., AAC 2011

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Plasmid pKP3-A carrying blaOXA-181

OriV mobC

mobA

mobB

mobD

ISEcp1 blaOXA-181

∆lysR

∆ere

repA

oriV

pKP3-A

7,605 bp

A ColE-type mobilizable and broad-host range plasmid

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blaOXA-181

Desoxyribonuclease Shewanella sp. 96%

Hyp. protein Shewanella sp. 92%

Hyp. protein Shewanella sp. 96%

lysR Carbamoyl-phosphate synthase Shewanella sp. 99%

100% identity

blaOXA-181 ISEcp1 ∆lysR

∆ere

Kp 481814

The blaOXA-181 originates from Shewanella xiamenensis

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And what about the famous blaNDM-1 gene ?

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The blaNDM-1 gene is part of composite transposon Tn1 25 in A. baumannii

blaNDM-1

ISAba125

IRL IRR

bleMBL iso tat dct groES groEL ISCR21

ISAba125

IRL IRR

∆pac

oriIS

2 copies of ISAba125

•ISAba125 belongs to the IS30 family, and encodes a 322 amino-acid long transposase

Poirel et al., AAC in press

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Transposon Tn1 25

blaNDM-1

ISAba125

IRL IRR

bleMBL iso tat dct groES groEL ISCR21

ISAba125

IRL IRR

∆pac

oriIS

Prom

ISAba125 provides the -35 box constituting the blaNDM-1 promoter

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Analysis of a blaNDM-1-carrying plasmid in an E. coli isolate

p271-A

re

p271

ccgA

I

mpr

ardK

stbC

Locu

s Tra

korB

blaN

DM

-1

kikA

stbB

st

bA

ISEc

33

tnpR

Locu

s Tra

KJI

tnpA

oriT

∆fip

A

reso

lvas

e

ssb

tnpA

nuc

GFOENDCBAM L

korA

Locu

s Tra

stbA

KJI

nuc

IS26

blaK

PC-3

tnpA

tnpA

oriT

P12

tnpA

tn

pA

IS4

rec

aacA

4 re

s Tn3

aa

dA1

blaO

XA-9

bl

aTEM

tn

pA

∆tnp

A ap

h sul1

∆fip

A tn

pA

tnpA

tn

pA

EcoR

II

korB

ki

kA

GFOENDCB

korA

AM L

arsC

ar

sB

arsA

ar

sD

arsR

tnpA

IS61

00

intI1

df

rA14

tnpR

tn

pM

∆Loc

us T

ra

IncN

rep

ccgA

I cc

gAII

mpr

muc

A m

ucB

ardK

stbC

ardR

ar

dB

stbB

ccgE

III

reso

lvas

e

ccgA

I

stbC

st

bB

IncN

rep

ccgC

ccgE

III

mpr

muc

A m

ucB

ardK

ardR

ar

dB

ccgD

P9

EcoR

II

stbA

KJI

nuc

fipA

ISEc

8 ex

cA

pndR

Φin

t tn

pA

tnpA

bl

aKPC

-2

IS4

tnpA

IS

4

blaK

PC-2

tnpA

tn

pA

Φin

t

oriT

reso

lvas

e

korB

ki

kA

korA

orfD

orfD

orfD

GFOENDCBAM L

ISSe

n4

Tn50

1 ISAb

a125

CAGGGGTC CAGGGGTC

A plasmid possessing an IncN-type scaffold, but with a new replicase

blaNDM-1

blaKPC-3

blaKPC-2

Poirel et al., AAC 2011

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blaNDM-1 IRL ISEc33 IRR IRL IRR ISSen4 IRL bleo

Close genetic environment of blaNDM-1

•Two novel insertion sequences are bracketing the blaNDM-1 gene

•ISEc33 has interrupted insertion sequence ISAba125

•No target site duplication, two different IS => likely not a composite transposon

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ISAba1 25 provides part of the promoter sequences for blaNDM-1 expression

TCAGATCGCACAATCCGGGC TGAGTTCCCTTTCAGTGAT CTACTATTCCGCGCAGCTAT TTAGAAAGGCGTTAGATTGGC TTACACCATTAGAGAAA IRL ISEc33 TTTGCTCAGCTTGTTGATTA TCATATGGCTTTTGAAAC TGTCGCACCTCATGT TTGAAT TCGCC CCATATTTTTGC TACAGT GAACCAAATTA AGAT IR L ISAba125 -35 -10 CATCTATTTACTAGGCCTCG CATTTGCGGGGTTTTTAATG CTGAATAAAAGGAAAACTTG ATGGAATTGCCCAATATTATGCA CCCGGTCGCG -> bla NDM-1

Promoter

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blaNDM-1 IRL ISEc33 IRR IRL IRR ISSen4 IRL bleo

A bit further…

CAGGGGTC

∆ISAba125 tnpR tnpA

CAGGGGTC

∆Tn501

=> A Tn501-made transposon

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blaNDM-1 IRL ISEc33

A.

B.

IRR IRL IRR ISSen4

blaNDM-1 ∆Tn3 ∆IS26 efflux pump ∆ldh ∆blaDHA-1 ∆PAI

ISAba125

∆ISAba125

C.

D. blaNDM-1

blaNDM-1

IRL bleo

bleo

bleo

bleo

ISAba125 blaNDM-1

E.

blaNDM-1 gene environment among different Enterobacteriaceae

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blaNDM-1 IRL ISEc33

A.

B.

IRR IRL IRR ISSen4

blaNDM-1 ∆Tn3 ∆IS26 efflux pump ∆ldh ∆blaDHA-1 ∆PAI

ISAba125

∆ISAba125

C.

D. blaNDM-1

blaNDM-1

IRL bleo

bleo

bleo

bleo

The same promoter is always present

Prom

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Conclusions

• A diversity of genetic tools for carbapenemase genes acquisition and dissemination is identified

• Genetic plasticity at the origin of certain carbapenemase gene diffusion => increasing spread

• Searching for the reservoirs Reservoir of the resistance genes themselves ? Reservoir of the mobilizing elements ? Which are the factors enhancing the mobilization

processes ?

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