ESCMID Online Lecture Library. by author

Timothy R. Walsh

Genetic Elements that spread multi-drug resistance determinants in Gram-negative bacteria:

how complex is the problem?

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Remit
of
Talk

• Will
not
detail
integrons
and
movement
of integrons

• Will
choose
few
and
only
key
examples
of 1.
plasmids

2.
transposons

3.
ISCR
elements...
to
illustrate
clinically import
examples
–
and
choose
key
resistant genes

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Channel Four Study

171 swabs

-156 grew meropenem resistant Gram-negatives)

- 51/171 (29.8%) were positive for NDM-1

50 water samples

- 14 grew meropenem resistant Gram-negatives)

- 2 out of 50 (4%) were positive for NDM-1

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3 97 116

Environmental positive samples

Escherichia coli

Suttonella indologenes Shigella boydii

Stenotrophomonas maltophilia Aeromonas caviae

Citrobacter freundii

Pseudomonas oryzihabitans

Pseudomonas pseudoalcaligenes Pseudomonas putida

Vibrio cholera ESCMID Online Lecture Library

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Walsh et al., Lancet Infectious Diseases, April 2011

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Walsh et al., Lancet Infectious Diseases, April 2011

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bla _NDM-1 genes are carried on AC plasmids

Some isolates have two copies of bla ESCMID Online Lecture Library

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P T P T P T P T M P T P T P T P

T ESCMID Online Lecture Library

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–Changes
to
NDM‐1
A/C
plasmids
during
conjuga;on


–P T P T P T P T –IR9



IR9





IR19



IR22 –P T P T P T P T –IR3




IR9




IR19



IR22

–180kb –160kb –150kb

–90kb

–F –A/C

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Transposons, also called "hopping genes" are

segments of DNA that are able to move around in the genome

They were originally suggested by Barbara

McClintock based on research she did the 1930's and 1940's

The simplest kind code only for the enzymes that cut them out of their current DNA molecule and insert them into another one

What are Transposons?

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What
are
IS
Elements

• Small
and
simple...generally
encode
no
funcCons
other than
those
involved
in
their
mobility

• Required
in
cis,
in
par,cular
recombina,onally
ac,ve

• DNA
sequences
which
define
the
ends
of
the
element, together
with
an
enzyme,
the
transposase
which

recognizes
and
processes
these
ends

• The
transposase
is
generally
encoded
by
one
or
perhaps two
open
reading
frames
and
consumes
nearly
the
enCre length
of
the
element

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Mechanism of transposition

• The simplest sequence of events:

• The transposase recognizes and binds to the transposable element

• It then acts as a site-specific endonuclease, cleaving the DNA to expose the element's 3'-OH ends

• A duplex target DNA then binds to the complex

• Direct attack of the 3'-OH ends on phosphates of the duplex cleaving it and joining it to the transposon

• Since the phosphates attacked are staggered, short single strand gaps remain, which are filled in by the regular repair mechanisms

• Mode of binding is the same as the mode of binding of transcription factors to DNA: a helix in the major groove.

• Some Type 1 transposases cleave the 5' ends of the transposon as well.

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DNA
Binding Domain
of
Tn3 Transposase Bound
to
the Transposon Tn5
Bound

to
Ends
of Transposon

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Nomenclature of Transposons

Revised
nomenclature
for
transposable gene,c
elements

Chandler et al., 2008. Plasmid, 60; 167-173

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INT aacA7 bla_VIM‐2 aacC1 aacA4 qacEÄ1/sul

French VIM-2

INT bla_VIM‐2 aacA4 qacEÄ1/sul

Poland VIM-2

GeneCc
Elements
changing
and
moving
resistance genes
around
Europe

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Schematic representation of the blaIMP-13 and blaVIM-2 genetic loci, and comparison with the 5′ end of the composite mercury resistance transposon Tn21, which includes the

transposition (tnp) and integron In2 region (GenBank accession no.

Toleman M A et al. J. Antimicrob. Chemother. 2003;52:583-590

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TGTCGTTTTCAGAAGACGGCTGCAC---AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG TGTCGTTTTCAGAAGACGGCTGCAC---AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG TGTCGTTTTCAGAAGACGGCTGCAC---AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG TGTCGTTTTCAGAAGACGGCTGCAC---AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG TGTCGTTTTCAGAAGACGGCTGCAC---AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG TGTCGTTTTCAGAAGACGGCTGCAC---AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG TGTCGTTTTCAGAAGACGGCTGCAC---AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG TGTCGTTTTCAGAAGACGGCTGCAC---AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG TGTCGTTTTCAGAAGACGGCTGCAC---AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG TGTCGTTTTCAGAAGACGGCTGCAC---AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG TGTCGTTTTCAGAAGACGGCTGCAC---AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG TGTCGTTTTCAGAAGACGGCTGCAC---AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG

*

IRi IRi

Tn5090/Tn402‐like

IRt

Δtnib


tniA qacEΔ1/sul

aadA2 aadB

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Evolution of Tn5090/Tn402-like elements

Toleman et al., 2007. Antimicrob Agents Chemother. 51 2636-8. ESCMID Online Lecture Library

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Representation of Tn4401a harbouring blaKPC -3 and the PCR scheme used to amplify it.

Curiao T et al. J. Antimicrob. Chemother. 2010;65:1608 -1614

© The Author 2010. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e -mail:

[email protected]

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Tato et al., 2010. ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 54; 320–327

ESCMID Online Lecture Library

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Tato et al., 2010. ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 54; 320–327 ESCMID Online Lecture Library

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Lar,gue
et
al.,
2004.
FEMS.
234:
201‐207 ESCMID Online Lecture Library

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ISEcp1B
mediated
transposiCon

Poirel et al., 2005. AAC. 49:447-550 ESCMID Online Lecture Library

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What
are
ISCR
Elements?

• One
ended
transposiCon
elements

• Only
recently
been
associated
with
anCbioCc resistance

• Replicate
via
rolling
circle
transposiCon

• Capable
of
carry
large
segments
of
DNA
to their
leX
hand
side
including
many
anCbioCc resistance
genes

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qac/sul qac/sul orf5

aadB dfrA10

L06418
(In7)

qac/sul

aadA2 catA2 qac/sulorf5/orf6

L06822
(In6) AJ517791

Common Regions - 1993

Common Region

Stokes HW, Tomaras C, Parsons Y, Hall RM. Plasmid. 1993, 30:39-50 ESCMID Online Lecture Library

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Direc&on
of
rolling
circle
replica&on


co‐transposed
genes

ISCR5 oriIS

terIS

bla_OXA-45

IS91 oriIS

terIS

eltA/B

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qacEΔ1/sul

aadA2 orf5/orf6

terIS oriIS

Deletion event removing terIS and fusing the ISCR1 element to the 3’ conserved sequence of the class 1 integron

oriIS intI1

qacEΔ1/sul aadA2

intI1

ISCR1

ISCR1

FormaCon
of
Complex
Class
1
integrons

ESCMID Online Lecture Library

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Coque
and
Canton.
Current
Opinion
in
Microbiology.
2006. ESCMID Online Lecture Library

© by author

oriIS

dfrA10 dfrA10 dfrA10

aadB orf5/orf6 aadB orf5/orf6 aadB orf5/orf6

aadB dfrA10 orf5/orf6

A

A B

aadB dfrA10 orf5/orf6

B

C

C

dfrA10

(i) (ii) (iii)

(iv) (v) (vi)

(i) (ii) (iii) qacEΔ1/sul1

aadA2 intI1

qacEΔ1/sul1 aadA2

qacEΔ1/sul1

dfrA10 qacEΔ1/sul1 dfrA10 aadA2 qacEΔ1/sul1

qacEΔ1/sul1 aadA2

intI1

qacEΔ1/sul1 aadA2

intI1

qacEΔ1/sul1 qacEΔ1/sul1

qacEΔ1/sul1

ISCR1 ISCR1 ISCR1

ISCR1 ISCR1

ISCR1

ISCR1

ISCR1

ISCR1

ISCR1 ISCR1

ISCR1

Toleman
et
al.,
JAC.
2006 ESCMID Online Lecture Library

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Toleman
et
al.
2007.
Emerging
Infec,ous
Diseases.
13;
559‐565 ESCMID Online Lecture Library

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SGI1

qac/sul floR qac/sul

bla_PSE‐1

tetG qac intI1/groEL orf5

aadA2

SGI1‐A

qac/sul floR tetG qac intI1/groEL qac/sul dfrA10 qac/sul orf5

aadA2

qac/sul

aadA2 dfrA10 qac/sul orf5

SGI1‐D

qac/sul qac tetG floR

IS6100 intI1/groEL

orf5/6 qac/sul floR

aadA2

IS6100 SGI1‐E

floR

qac/sul tetG qac intI1/groEL qac/sul orf5/6

aadA2

dfrA1 orfC

SGI1‐I qac/sul

dfrA1 orfC floR tetG qac intI1/groEL qac/sul

SGI1‐F

qac/sul

dfrA1 orfC floR tetG qac intI1/groEL qac/sul

SGI1‐J

erm

A15097 AY434092 yieF/yieG

AY434093 yieE

yieE/yieF/orfX

AY434091 bla_PSE‐1

bla_PSE‐1

bla_PSE‐1

Scale
=1kb

intI1/groEL bla_TEM‐1 rmtB

tnpR AB103506

Importance of ISCR3 elements (55%ID ISCR1)

I^SCR3

Toleman
et
al.
2006.
Microbiology
and
Molecular
Biology
Reviews.
70:
296‐316.

hhp://www.cardiff.ac.uk/medic/aboutus/departments/medical microbiology/geneCcs/iscrelements.html

ESCMID Online Lecture Library

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•A class of bacterial mobile genetic elements that disseminate via conjugation and then integrate into the host cell genome.

•The SXT/R391 family of ICEs consists of more than 30 different elements that all share the same integration site in the host chromosome but often encode distinct properties.

•Comparative analyses of the genomes of several SXT/R391 ICEs, found that the genomes have been shaped by inter–ICE recombination.

•Conjugation facilitates the segregation of hybrids and could provide a means to select for functional recombinant ICEs containing novel combinations of genes conferring resistance to antibiotics.

•ICEs promote their own diversity and can yield novel mobile elements capable of disseminating new combinations of antibiotic resistance genes.

Integrating conjugative elements (ICEs)

Genevie`ve

et
al.,
2009.
PlosGen
5;
1‐11 ESCMID Online Lecture Library

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How
complex?

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Fournier
et
al.
2006.
Compara,ve
genomics
of
mul,‐drug
resistance
in
Acinetobacter
baumannii.

Gene,cs.
2:
62‐72 ESCMID Online Lecture Library

© by author

Fournier
et
al.
2006.
Compara,ve
genomics
of
mul,‐drug
resistance
in
Acinetobacter
baumannii.

Gene,cs.
2:
62‐72

AnCbioCc
genes
belonging
to
MDR
A.
baumannii
strain
AYE

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(4-6kb)

(1-4kb)

Yong et al., 2009. Antimicrobial Agents and Chemotherapy. 53; 5046-54 Poirel et al. 2010. Antimicrobial Agents and Chemotherapy. 54; 4914-4916

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ISCR NDM-1 ISCR groEL groES NDM-1

Gene X

...

AbaI1 25

NDM-1 Gene X

...

∆AbaI1 25

The perfect weapon?

DHA-1, CMY-6, aar-2, ereC, aadA1, aacA4, cmlA7, armA, rmtC, rmtD, sul1, dhfr, sul2, toxin-anti-toxin system - MqsA

Curr
Opin
Microbiol.

2010
Dec;13(6):781‐5.

Toxin‐an,toxin
systems:

why
so
many,
what
for?

Van
Melderen
L . Acinetobacter spp.

Enterobacteriaceae

Enterobacteriaceae

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Event
1 Event
2 Event
3 Event
4

Chronology
of
movement
of
AbxR
genes

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Major structural features of pKOX105, encoding VIM -1, QnrS1 and SHV -12, in comparison with IncN plasmids p9 and p12 carrying KPC -2 and KPC -3, respectively, and the IncN reference

plasmid R46.

Carattoli A et al. J. Antimicrob. Chemother. 2010;jac.dkq269

© The Author 2010. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e -mail:

[email protected]

ESCMID Online Lecture Library

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How complex is the problem?

• Although
known
for
a
long,
transposons
have
only recently
been
associated
with
MDR
and
PDR
Gram‐

negaCve
bacteria

• Safe
haven
for
inserCon
but
can
also
be
destrucCve

• This
associaCon
may
or
may
not
be
associated
with drug
consumpCon
but
anCbioCcs
such
as

fluoroquinolones
have
secondary
effects
promoCng gene
mobility

• The
complexity
(and
associated
plasCcity
and
fluidity) will
become
greater
over
Cme

• A
warning
to
genome
sequencers

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Acknowledgements

• Mark Toleman

• Dongeun Yong

• Sonia Ferreira

• Allaaeddin El Salabi

• Sahim Agouri

• Jonathan Tynddell

• Janis Weeks

• Mandy Wootton

• Robin Howe

• Vicky Davies

• Laurent Poirel

• Patrice Nordmann

• Jan Bell

• David Paterson

• Hanna Sdajbat

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