Alessandra CarattoliIstituto Superiore di Sanità, Roma IT

Public health perspectives on antimicrobial resistance plasmids in Enterobacteriaceae

Molecular epidemiology of antimicrobial resistance: the

targets• AR gene

• transposon

• plasmid

• bacterial clone

replication stability conjugation resistance mobile elements other genes

Rep

licas

e

Inse

rtion

sequ

ence

Parti

tioni

ngR

estri

ctio

nen

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

Small (<30 kb) mobilizable plasmidoriV oriT

Inse

rtion

sequ

ence

mob

A/B

mob

C

repA

/B/C

itero

ns

Res

ista

nce

Inse

rtion

sequ

ence

oriV

Resistance

objective:to trace resistance plasmids

PLASMID TYPING AND SUBTYPING

1st level: PCR

PCR Multiplex(bp)

PCR Simplex(bp)

1 2 3 4 5

HI1471

HI2644

I1139

X376

L/M785

N559

FIA462

FIB702

W242

Y765

P534

FIC262

A/C465

T750

FIIS270

FII270

K155

B/O147

PCR-Based Replicon Typing (PBRT):plasmids are detected using replicase genes as targets

PBRT-KIT

M1

•HI1 (534)•HI2 (308)•I1α (159)

M2

•M (741)•N (514)•I2 (316)•B/O (159)

M3

•FIB (683)•FIA (462)•W (242)

M4

•L (854)•P (534)•X3 (284)•I1γ (161)

M5

•T (750)•A/C (418)•FIIS (259-260)

M6

•U (843)•X1 (370)•R (251)•FIIK (142-148)

M7

•Y (765)•X2 (376)•FIC (262)•K (160)

M8

•HIB-M (570)•FIB-M (440)•FII (258-262)

objective:to trace resistance plasmids

PLASMID TYPING AND SUBTYPING

2nd level: PCR-sequencing of the amplicon

pDLST(plasmid double locus sequence typing)

smr 0018 (1118 bp)smr 0199 (536 bp)

IncHI2 (multiplex PCR)

hipAsmr 0092smr 0183

repI: replicase gene

ardA: type I restriction-modification enzyme

trbA: involved in maintenance and plasmid transfer

sogS: DNA primase

pilL: type IV pilus biogenesis

IncI1 IncHI2

FII: The copA region of the FII replicon FIA: The region comprising the iterons and the replication protein repE gene of the FIA replicon FIB: The replication protein repB gene of the FIB replicon FIC: The copA region of the FIC replicon

IncF IncN

repN: replicase gene traJ: mobilization of the plasmid during the conjugative transfer korA: negatively regulating the synthesis of the conjugal pilus and mating pore during the conjugation process

García-Fernández et al. 2011Villa et al. 2010

García-Fernández & Carattoli. 2010García-Fernández et al. 2008

IncHI1 HCM1 043; HCM1 064; HCM1 099; HCM1 116; HCM1 178ac; HCM1 259 Phan MD& Wain J

Plasmid MultiLocus Sequence Typinghttp://pubmlst.org/plasmid/

objective:to trace resistance plasmids

PLASMID TYPING AND SUBTYPING

3rd level: whole genome sequencing (WGS)

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

I1 plasmids and ESBLs

Plasmid transmission among bacteria from animals and humans

avian commensal E. coli (AFEC) avian pathogenic E. coli (APEC) retail poultry E. coli (RPEC)human commensal E. coli (HFEC) uropathogenic E. coli (UPEC)

Replicon AFEC %(n=92)

APEC %(n=422)

RPEC %(n=200)

HFEC %(n=101)

UPEC %(n=200)

I1 17.4 41.0 34.5 6.9 6.5

Occurrence of IncI1 plasmid replicons among various E. coliisolates- PBRT

Johnson TJ et al.,Plasmid Replicon Typing of Commensal and Pathogenic Escherichia coli Isolates. 2007 AEM 73: 1976–83

pMLST identified related IncI1 plasmids in isolatesfrom animal and human sources

CC‐3, CC‐7: CTX‐M‐1

CC‐12: CMY‐2

CC‐5: TEM‐52 TEM‐20

CC31: CTX‐M‐15

CC‐3, CC‐7: CTX‐M‐1Leverstein-van Hall et al CMI 2011

The birth of a resistance gene:CTX-M comes from Kluyvera

Rodríguez M M et al. Antimicrob. Agents Chemother. 2004;48:4895-4897

CTX-M

0

14.7

2468101214

CTX-M-43 (DQ102702)Toho-1 (D37830)CTX-M-31 (AJ567481)CTX-M-35 (AB176532)CTX-M-2 (X92507)CTX-M-20 (AJ416344)CTX-M-4 (Y14156)CTX-M-7 (AJ005045)CTX-M-6 (AJ005044)KLUA-5 (AJ427463)KLUA-12 (AJ427469)KLUA-4 (AJ427462)KLUA-3 (AJ427461)KLUA-1 (AJ272538)KLUA-8 (AJ427465)KLUA-9 (AJ427466)KLUA-11 (AJ427468)CTX-M-5 (U95364)KLUA-6 (AJ427464)KLUA-2 (AJ251722)KLUA-10 (AJ427467)CTX-M-25 (AF518567)CTX-M-39 (AY954516)CTX-M-26 (AY157676)CTX-M-41 (DQ023162)CTX-M-8 (AF189721)KLUG-1 (AF501233)CTX-M-40 (AY750914)CTX-M-1 (X92506)CTX-M-32 (AJ557142)CTX-M-36 (AB177384)CTX-M-29 (AY267213)CTX-M-30 (AY292654)CTX-M-11 (AJ310929)CTX-M-33 (AY238472)CTX-M-15 (AY044436)=UOE-1CTX-M-28 (AJ549244)CTX-M-23 (AF488377)CTX-M-42 (DQ061159)CTX-M-22 (AY080894)CTX-M-3 (Y10278)CTX-M-12 (AF305837)CTX-M-10 (AF255298)CTX-M-34 (AY515297)CTX-M-37 (AY649755)KLUC-1 (AY026417)CTX-M-38 (AY822595)Toho-2 (D89862)CTX-M-19 (AF325134)CTX-M-24 (AY143430)CTX-M-9 (AF174129)CTX-M-16 (AY029068)CTX-M-27 (AY156923)CTX-M-14 (AF252622)=M-18=Toh3=UOE-2CTX-M-17 (AY033516)CTX-M-13 (AF252623)CTX-M-21 (AJ416346)

pMLST identified related IncI1 plasmids in isolatesfrom animal and human sources

CC‐3, CC‐7: CTX‐M‐1

CC‐12: CMY‐2

CC‐5: TEM‐52 TEM‐20

CC31: CTX‐M‐15

CC‐3, CC‐7: CTX‐M‐1Leverstein-van Hall et al CMI 2011

The travels of CTX-M-1 and CTX-M-15

CC7-CTX-M-1CC3-CTX-M-1 CC31-CTX-M-15

Characterization of Epidemic IncI1-I Plasmids Harboring Ambler Class A and C Genes in Escherichia coli and Salmonella entericafrom Animals and Humans. H. Smith et al., 2015, AAC 9:5357-65

pMLST-I1 plasmid validation by WGS

Another example:diversity of plasmids at the origin of dissemination of NDM-1 carbapenemase

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

The carbapenemase threat in the animal world: the wrong culpritPoirel L, et al., J Antimicrob Chemother. 2014 Jul;69(7):2007-8.

NDM-type carbapenemase dissemination

EARSS 2015Escherichia coliResistance to carbapenems R+I

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

Escherichia coliKlebsiella pneumoniaeProvidencia stuartii

Identification of NDM-positive IncA/C plasmids. A. Carattoli 2016, unpublished blaNDM-1 + armA + blaCMY-16

Milvus migransfosA3

fosphomycin resistancesuspected

origin in Asiatic regions

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

Plasmids without frontiers• MCR-1: plasmid mediated colistin resistanceAnimal reservoir?Human origin?Food-related transmission?Hospital, community, travels?

Species Source Country Year Beta-lactamases Plasmid Reference

E. coli Poultry ChinaDenmarkFranceAlgeriaMalaysia

2005-2015 CTX-M-1CMY-2 SHV-12

I2X4 HI2NT

Liu et al., LID 2016Hasman et al., EurSurv 2015Olaitan et al., LID 2016Petrillo et al., LID 2016Perrin-Guyomard et al., EurSurv 2016

E. coli Cattle FranceBelgiumJapan

2005-2015 CTX-M-1CTX-M-27

I2PHI2NT

Haenni et al., LID 2016Suzuki et al., LID 2016Malhotra-Kumar et al., LID 2016Perrin-Guyomard et al., EurSurv 2016

E. coliSalmonella

Swine ChinaBelgiumGermanyLaosMalaysiaJapanVietnam

2010-2013 CTX-M-1CTX-M-27CTX-M-55

I2HI2 X4NT

Liu et al., LID 2016Olaitan et al., LID 2016Petrillo et al., LID 2016Falgenhauer et al., LID 2016Suzuki et al., LID 2016Malhotra-Kumar et al., LID 2016Perrin-Guyomard et al., EurSurv 2016

E. coliSalmonella

Food ChinaSwitzerlandPortugalFrance

2011-2014 CTX-M-55CTX-M-65NDM-9

I2P X4NT

Liu et al., LID 2016Tse et al., LID 2016Petrillo et al., LID 2016Webb et al., LID 2016Malhotra-Kumar et al., LID 2016Yao et al., LID 2016Zurfuh et al., AAC 2016

E. coli Water SwitzerlandMalaysia

2012-2013 SHV-12 NT Petrillo et al., LID 2016Zurfuh et al., AAC 2016

More MCR-1 findings in animals, food, environment

Species Country Year Beta-lactamases Plasmid Reference

E. coli K. pneumoniae

ChinaDenmarkSwitzerlandTunisiaCambodiaPerùBoliviaGermanyLaosThailandiaVietnam

2012-2015 CMY-2VIM-1KPC-2CTX-M-1CTX-M-14CTX-M-15CTX-M-65CTX-M-55

I2HI2NT

Liu et al., LID 2016Hasman et al., EurSurv 2015Poirel et al., LID 2016Arcilla et al., LID 2016Stoesser et al., LID 2016Falgenhauer et al., LID 2016Olaitan et al., LID 2016Du et al., LID 2016

More MCR-1 findings in humans

Map of IncI2 plasmid pHNSHP45 carrying mcr-1 from Escherichia coli 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

IncI2 plasmid associated with mcr-1

Horizontal transmissionMultifocal bacterial strainsSuccessfull broad-host range plasmidsEpidemic plasmids