Alessandra CarattoliIstituto Superiore di Sanità, Rome Italy

Plasmids and Antibiotic Resistance: Novel Insights into Bacterial Sex

Plasmids are genomes By conjugation, they circulate among bacteria of different species and generaThey control their replicationThey control their stabilityPlasmids can kill bacteria

Plasmids infect bacteria

replication stability conjugation resistance mobile elements other genes

Rep

licas

e

Inse

rtion

sequ

ence

Parti

tioni

ngR

estri

ctio

n en

zym

e

reso

lvas

e

Self-conjugative systemRes

ista

nce

gene

Toxi

n-an

titox

in

Met

hyla

se

Large (30-300 kb) self-conjugative plasmid

oriV

Resistance

Ilangovan et al., 2015 Structural biology of the Gram-negative bacterial conjugation systems, Trends in Microbiology, 23:301–310

KPC-producing Klebsiella pneumoniae

90

188

382

307

258

23

Diversity, virulence, and antimicrobial resistance of the KPC-producingKlebsiella pneumoniae ST307 cloneVilla L, C Feudi, D Fortini, S Brisse, V Passet, C Bonura, A Endimiani, C Mammina, AM Ocampo, JN Jimenez, Ml Doumith, N Woodford, K Hopkins and A CarattoliMicrobial Genomics , 2017, 3doi: 10.1099/mgen.0.000110

The role of epidemic resistance plasmids and international high-risk clones in the spread of multidrug-resistant Enterobacteriaceae.Mathers AJ, Peirano G, Pitout JD.Clin Microbiol Rev. 2015 Jul;28(3):565-91. doi: 10.1128/CMR.00116-14.

Population Genomic Analysis of 1,777 Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae Isolates, Houston, Texas: Unexpected Abundance of Clonal Group 307. Long SW, Olsen RJ, Eagar TN, Beres SB, Zhao P, Davis JJ, Brettin T, Xia F, Musser JM. MBio. 2017 8(3). pii: e00489-17

Plasmid content of the K. pneumoniae high-risk clone ST258 producing KPC-3

ColEST258, Italy, 13640 bp, JN247853

Tn3

tnpA

aac6

moB

Tn3

tnpR

M-tr

ansf

cloa

cin

moC

ropB relE

IS26

locus tralocus mer

stbA

bla T

EM-1

parA

Tn3t

npA

Type

Ires

trict

ion

umuC

umuD

ardB

IS26IS

1

parB

stbBss

bpa

rB

ardA

Tox-

antit

ox

aphA

1a

tnpA

FIIK

2

tnpA

Tn3

tnpR

ISK

pn6

bla K

PC-3

ISK

pn7

help

ISK

pn7

TnpA

Tn44

01 tn

pA

Tn44

01 tn

pR

FIBK

QIL

Type

IIIre

stric

tion

TniA

tnpA

IS26

pKpQIL, Italy, 114986 bp, JN233705

García-Fernández et al., A

ntimicrob A

gents Chem

other. 2012 ; 56:2143-5

KPC-3

Tn4401

macrolides, trimethoprim, sulphonamides, streptomycin, chloramphenicol

Arsenic, Cupper and Silver

gentamycin

replication stability conjugation resistance Mobile elements virulencecarbapenemase

Mercuric ions, kanamycin

pKPN3 ST258, 208193 bp (JN233704)

Fe(3

+) u

ptak

e

copp

er/s

ilver

IS90

3

dfrA

12

IS43

21

aadA

2

FII K

1 IS1

IS26

sul1

tnpA

Tn21 mph

A

IS1

IS20

4

arse

nic IS1

IS90

3FI

B K

PN

umuC

lacY

, Z, I

tnpA

intI1

sopAIS

1

sopB

umuD

Rev

Tran

smrx

cat

IS61

00

ABC

-glu

tath

ione

IS43

21

tnpR

Tn21

Lactose fermentationLacY, LacZ, LacI

Iron uptakeFe(3+) aerobactin

glutathione importation

locus tra

Plasmid content of the K. pneumoniae high-risk clone ST307 producing KPC-3 Villa et al., M

icrob Genom

. 2017 Apr 26;3(4):e000110.

pKPN3 ST307, 227989 bp (KY271404)

Gly

coge

n sy

nthe

sis

bla C

TX-M

-15

bla T

EM-1

Fe(3

+) u

ptak

e

ABC

-glu

tath

ione

ABC

-ure

a

copp

er/s

ilver

IS1

IS26

IS26

dfrA

14

ISEc

p1

IS43

21strB

IS43

21

tnpR

tnpA

Tn50

1qn

rB1

EcoR

II

FII K

7

IS61

00

intI1

tnpR

Tn5

01IS

26

bla O

XA

-1aa

c6’-1

b-cr

IS26

IS26

aacC

3IS

26tn

pATn

3

sul2 IS1

IS20

4

arse

nic

IS1

IS90

3FI

BK

PN

umuC

lacY

, Z, I

tnpA

IS43

21

sopAIS

1

sopB

umuDstrA

∆cat

B3

pKpQIL-ST307, 116499 bp (KY271403)

DN

A m

ethy

lase

bla K

PC-3

tnpA

ISK

pn7

FII K

2

ISK

pn6

Tn44

01 tn

pRTn

4401

locu

s mer

stbA

parA

ISK

pn25

Type

I res

trict

ion

umuC

umuD

ardB

IS26 IS

1

parB

stbB ss

bpa

rB

ardA

Tox-

antit

ox

rest

r. En

donu

cl. S

sub

Tn3

tnpA

tnpR

FIBK

QIL

Type

III re

stric

tion

Tn2

tnpA

IS26

bla T

EM-1

tnpR

bla O

XA

-9

KPC-3

Tn4401

Mercuric ions

Lactose fermentation

Iron uptakeFe(3+) aerobactin

Glutathione importation

Glicogen synthesisUrea transporter

replication stability conjugation resistance Mobile elements virulencecarbapenemase

ESBL (CTX-M-15), Cip, Gen, Amk, Str, Tmp, Sul, Ars, Cu, Si

Type A ICE (Group VI), 64533 bp (KY271411)

Type B ICE (Group VI-like), 58182 bp (KY271412)

tRNA-Asn1A tRNA-Asn1B tRNA-Asn1C tRNA-Asn1D

Klebsiella pneumoniae CAV1193 (CP013322; nt. 4088248..4066852)

yersiniabactin Type IV SS

int R M

AB

C

hyp

ardC

mob

Cm

obB

kikA hy

p

hyp

hyp

hyp

hyp

hyp

int

hyp

hyp

hyp

kikA hy

pm

obB

mob

Car

dC R hyp

hyp

hyp

hyp

hyp

RT hyp

tnpA

yersiniabactin Type IV SS

Villa et al., M

icrob Genom

. 2017 Apr 26;3(4):e000110.

Emergence and rapid global dissemination of CTX-M-15-associated Klebsiella pneumoniae strain ST307. Wyres KL, Hawkey J, Hetland MAK, Fostervold A, Wick RR, Judd LM, Hamidian M, Howden BP, Löhr IH, Holt KE. JAC. 2018 Dec 4. doi: 10.1093/jac/dky492

Geographical distribution and increasing reports of ST307

Emergence and rapid global dissemination of CTX-M-15-associated Klebsiella pneumoniae strain ST307. Wyres KL, Hawkey J, Hetland MAK, Fostervold A, Wick RR, Judd LM, Hamidian M, Howden BP, Löhr IH, Holt KE. JAC. 2018 Dec 4. doi: 10.1093/jac/dky492

Animal reservoir of AMR

pMLST databases

PlasmidFinder and pMLST in silico

http://cge.cbs.dtu.dk/services/PlasmidFinder/http://cge.cbs.dtu.dk/services/pMLST/

In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing A Carattoli et al., AAC 58: 3895-3903

http://www.iss.it/pres/prim/cont.php?id=1277&lang=1&tipo=6

http://pubmlst.org/plasmid/ (2018)

Plasmid Inc types in Escherichia coli pMLST isolate database

http://pubmlst.org/plasmid/

γ

2.1

2.2

1.11.2

2.3

2.4

γ

γ

CMY-2(CC-2)

CTX-M-1(CC-7)

CTX-M-1(CC-3)

TEM-52(CC-5)

CTX-M-15 (CC-31)

CMY-2(CC-12)

γ

Cluster I

Cluster II

pMLST MAFFT analysis of 291 STs for IncI1

CTX-M-15 (ST-16)

Cluster III

Carattoli et al., 2018 Plasmid in press

http://pubmlst.org/plasmid/ (IncI1 plasmids, Sept 2018)

Carattoli et al., 2018 Plasmid in press

http://pubmlst.org/plasmid/

ESBLAmpC

Species Country Year Sources ST (CC) Clade Cluster

CTX-M-15

E. coli, Shigella sonneiAustralia, Belgium, France, Germany, The Netherlands, UK, USA

2001-2011human, horse, cattle

31 (CC-31) 1.2 I

Salmonella Typhimurium, Salmonella Anatum

UK 2001-2003 human 8 (CC-31) 1.2 I

E. coli, Shigella sonnei Ireland, South Korea, UK 2009 human, cattle 16 2.3 II

E. coliAustralia, Switzerland, The Netherlands, UK

2006-2009 human, cattle 37 2.3 II

CTX-M-1

E. coli, S. Newport, S. Typhimurium, S. Llandoff, S. London, S. Agona

Czech Republic, Denmark, France, Germany, Ireland, Italy, Poland, The Netherlands, UK

2005-2010human, poultry, pig, cattle, dog, other

3 (CC-3) 2.1 II

E. coli, S. Paratyphi B, S. Infantis, S. Agona

Germany, Ireland, The Netherlands

2006-2009human, poultry, pig, cattle

7 (CC-7) 2.1 II

E. coli Germany, Italy 2007 poultry, pig 26 (CC-26) 2.2 II

E. coli Germany 2009 cattle 63 2.4 II

TEM-52

E. coli, S. Infantis, S. Paratyphi B

The Netherlands 2006-2009human, poultry 10 (CC-5) 1.1 I

E. coli, S. Infantis, S. enterica, S. Paratyphi B

France, Germany, Switzerland, The Netherlands 2005-2018

human, poultry, cattle, pig

36 (CC-5) 1.1 I

CMY-2

S. Agona, S. Enteritidis, E. coli

Taiwan 2009-2010 human 56 1.1 I

E. coli, S. Heidelberg, S. 4,5,12:i:-, S. Typhimurium

Australia, Canada, Denmark, Italy, The Netherlands, UK, USA

2005-2009human, poultry, dog

2 (CC-2) 2.2 II

E. coli, S. Heidelberg, S. Newport

Canada, USA 2005-2009 human, dog 23 (CC-2) 2.2 II

S. Heidelberg, S. Typhimurium, S. Litchfield, E. coli, S. Paratyphi B, S. Newport

Brazil, Canada, Germany, The Netherlands, Uruguay, USA

2001-2013human, poultry, turkey

12 (CC-12) 2.4 II

S. Heidelberg USA 2009 human, poultry 66 (CC-12) 2.4 II

CMY-2:IncI1 CC-12ESBL and AmpC in animals:the most popular genes on IncI1

TEM-52:IncI1 CC-5CTX-M-1:IncI1 CC-3CTX-M-15-IncI1 CC-31

Mobile colistin resistance

Emergence of plasmid-mediated colistin resistancemechanism MCR-1 in animals and human beings in ChinaLiu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, Doi Y, Tian G, Dong B, Huang X, Yu LF, Gu D, Ren H, Chen X, Lv L, He D, Zhou H, Liang Z, Liu JH, Shen J.Lancet Infect Dis. 2016 Feb;16(2):161-8. Published online November 18, 2015

Colistin

Map of IncI2 plasmid pHNSHP45 carrying mcr-1 from Escherichia coli in China, 2016Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, Doi Y, Tian G, Dong B, Huang X, Yu LF, Gu D, Ren H, Chen X, Lv L, He D, Zhou H, Liang Z, Liu JH, Shen J. Lancet Infect Dis. 2016 Feb;16(2):161-8

IncI2 plasmid associated with mcr-1

pWJ1 IncHI2-type plasmid carrying mcr-3

Novel Plasmid-Mediated Colistin Resistance Gene mcr-3 in Escherichia coli.Yin W, Li H, Shen Y, Liu Z, Wang S, Shen Z, Zhang R, Walsh TR, Shen J, Wang Y.MBio. 2017 Jun 27;8(3). pii: e00543-17. doi: 10.1128/mBio.00543-17.

mcr-4.1

relE

excl1(pSC101)

repB(pSC101)

mobA (pSC101)

ISKpn2

0

1 kb2 kb

4 kb

5 kb 7 kb

pMCR-4.1_R34458749 bp

(MF543359.1)

mobA/LISKpn26 mcr-4.2repB excl1 relE ISKpn26

43 AntigenRD-RNA pol DNA helicase rloF ΔTypeI restriction TypeI restr. S TypeI restr. M ngrB klcA

Chromosomal integration of pMCR-4.2 (E.coli from pigs)

Magistrali CF, et al., Int J Antimicrob Agents. 2018 Aug 17. pii: S0924-8579(18)30234-6.

Carattoli A, et al. Eurosurveillance 2017;22(31):30589.

mcr-4.2

relE

excl1(pSC101)

IS1294

mobA (pMG8282)

repB(pMG8282)

0

3.2 kb 4.7 kb

1.6 kb 6.3 kb

7.9 kb

pMCR-4.2_AB-2439568 bp

(MG800340)

Carattoli A, et al., J Antimicrob Chemother. 2018 73(12):3332-3335.

Worldwide dissemination of the NDM-type carbapenemases in Gram-negative bacteria. Dortet et al., Biomed Res Int. 2014;2014:249856

NDM-type carbapenemase dissemination

pNDM-1 IncN2 Escherichia coli, Australia, 35947 bp , JF785549

repA ss

b

nuc

stbC stbB

stbA

∆fip

A

IS40

7bl

a ND

M-1

ISAb

a125

tnpR

tnpA

bleM

BL

pNDM-KN, IncA/C Klebsiella pneumoniae, Kenia, IncA/C 162746 bp, JN157804

ISEc

23

repA

/Cst

bA

nucl

ease

Type

I res

tric

Tn7

rmtC

cmlA

endo

nucI

II

intI1rifR

ereA

aadA

trhF

bla N

DM

-1

sul1

IS43

21

traC bl

cbl

a CM

Y-16

ISEc

p1

DN

Apol

IIID

NA

met

hyla

seD

NA

met

hyla

seko

rBpa

rA

ISK

pn14

DN

A m

ethy

lase

parBte

rya

cCph

age-

int

Δrhs

groE

Lgr

ESS

bleM

BL

hsdR

Tn16

96

pNDM-MAR, IncH-new Klebsiella pneumoniae, Morocco, 267,242 bp, JN420336

insKTn

2

trhA

dsbC

repF

IB

DN

A m

ethy

laseIS5

EcoR

II re

stric

tion

EcoR

II m

ethy

lase

dnaC

parA

parB

IS60

5

repH

IBIS

4321

Tn21

IntI1

IS26Tn

2IS

4321ca

tIS

4321 IS

1

Tn21Tn3

Locu

s tel

lurit

e

parB

trbN

tnpA

IS15

0

ISEc

p1IS

Ecp1

blaC

TX-M

-15

Tn3t

npA

IS26

blaO

XA

-1aa

c6-1

b

IS26Tn

3bl

a ND

M-1

bleM

BL

groE

LTn

3qn

rB1

IS26

groE

S

trhO

trhGtrhI

catB

3

insK

htdA

Ktr

hFrec

mer

R

IS66

dsbC

IS26

Tn17

21

pNDM-CIT, IncHI1 Citrobacter freundii, 288,920 bp, JX182975

EcoR

II m

ethy

l.

DN

A m

ethy

.

DN

A m

ethy

l.

repH

I1B

parB

parB

parAIS1

cat

dsbC trhI

repH

IA

prophage CP4

repA

ciN

htdV

T

htdA

FKtrhF

H-N

S lik

e

trhA

IS43

21

intI1

tnpA

Tn2

1tn

pR T

n21

dfrA

12qa

cEΔ1

ISC

R1

bla N

DM

-1bl

eMB

L

dfrA

12aa

dA2

ISC

R1

intI1

tnpD

arm

A

mph

2IS

26IS5

Ars

locu

s

tnpA

Locu

s tel

lurit

e

IS11

0IS

66IS

100

IS10

0

IS1

intA fic alpAIS

1

MDR efflux pump system

trbN

tnpU sul1

qacEΔ1mel

sul1

aadA

2

parR

parM

trhO htdO trhL

pNDM-GUE, IncF Escherichia coli, France, IncFII, 65245 bp , JQ364967

pNDM-HK IncM Escherichia coli, Honk-Kong, IncL/M 88,803 bp, HQ451074

repL

/Mbl

a TEM

-1IS

26

bla N

DM

-1bl

a DH

A-1

sul1

ISC

R1IS

Ec28

arm

AIS

Ec29

msr

E, m

phE

IS26

Tn2t

npA

IS18

6m

ucAB IS26

parA

parB ss

bm

obC

aacC

2IS

26

Locu

s Mer

replication stability conjugation resistance mobile elements other genes

Dolejska et al., J. Antimicrob. Chemother. 68 (1), 34-39 (2013)Villa et al., J. Antimicrob. Chemother. 67 (7), 1645-1650 (2012)Carattoli et al., Antimicrob. Agents Chemother. 56 (2), 783-786 (2012)Ho et al., PLoS ONE 6 (3), E17989 (2011)Bonnin et al., PLoS ONE 7 (4), E34752 (2012)Poirel et al. Antimicrob Agents Chemother. (2011) 55(9):4224-9

aacC

2

aadA

2IS

CR

1

dhfr

A12

intI1

repF

II

IS26

IS26

∆Tn

1721sul1

bla N

DM

-1

parBssb

parM

bleM

BL

catB

4bl

a OX

A-1

aacA

4IS

26

NDM

Plasmids without frontiers: IncA/C plasmids associated with NDM-1

Escherichia coliKlebsiella pneumoniaeProvidencia stuartii

NDM-positive IncA/C plasmids blaNDM-1 + armA + blaCMY-16

Milvus migrans

fosA3 fosphomycin resistance

suspectedorigin in Asiatic regions

Ho et al., JAM 2013China:cattle, pigs, chickens,dogs, cats, and rodents

•Persistance, stability and conjugation are offered by the plasmid to the resistance gene

•The success of a resistance gene may depend by the success of the plasmid

Laura Villa

Daniela Fortini

Istituto Superiore di SanitàDept. Infectious DiseasesAntibiotic Resistance UnitNational Reference Center for Enteric Pathogens

Claudia Lucarelli

Aurora Garcia-Fernandez

Anna Maria Dionisi

Slawomir Owczarek

Ildo Benedetti

Sergio Arena

Alessandra Carattoli

What does it mean “epidemic” plasmids?

1. Indistinguishable plasmids that have been detected in different countries, sources and bacterial species

2. Broad-host range plasmids that are able to replicate and conjugate at high efficiency

3. Plasmids carrying worldwide spread carbapenemase or plasmid-mediated colistin resistance genes

Which is the most important contribution of plasmids to the burden of antimicrobial resistance?

1. They are relevant because they are stably maintained within the bacterial cell

2. They are relevant because they are able to promote horizontal transmission of antimicrobial resistance genes among bacteria

3. They are relevant because plasmid DNA is stable and can survive without the bacterial host as naked DNA

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