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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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