Abstrack

Objectives
• Investigate the relevance of multicopy plasmids in AMR
and assess their mobilization by phage particles.
Results
• 60% of these plasmids do not bear mobility (MOB)
genes.
• mobilization of a marker gene armA  10000 times more
frequent on these multicopy plasmids.

Methods Conclusions
• The 16S methyltransferase gene armA used as a • Multicopy plasmids and phages deserve further
marker in eight different plasmids. investigations.
• All plasmids were transformed into Escherichia coli.
Introduction

• Little is known regarding the relationship between plasmids and phages.

• Many MCPs lack mobility (MOB) genes, but are widely spread in bacterial populations.

• Each plasmid is present in the cytoplasm in multiple copies  rise to 100 copies/cell during
antibiotic treatment.

• Antimicrobial resistance (AMR) genes normally found on plasmids have been identified in
phage particles.

• Like other bacterial DNA, plasmids can be transferred through generalized transduction.

• Things to prove the hypothesis  analysed the relevance of MCPs in bacteria, assessed
AMR genes present in MCPs, test the efficiency of phage encapsidation of an AMR gene
borne on different genetic platforms.
 Materials and Methods (1)
1. In silico analysis of plasmids in bacteria

• NCBI Nucleotide data base to see the

prediction of mobility  based on the
presence of relaxases and mating pair
formation systems using the MOB-typer
tool included in the MOB-suite software.

• The PLSDB web server for screening

purposes.

• Only plasmid records with circular

sequences or complete assemblies were
selected.

• Plasmids lacking a relaxase were

classified as ‘non-MOB’.

• The presence of a type IV secretion system Llosa, M., F. X. Gomis-Ruth, M. Coll, and F. de la Cruz.
(t4ss) ,involved in mating pair formation 2002. Bacterial conjugation: a two-step mechanism for
during conjugation, enabled us to separate DNA transport. Mol. Microbiol. 45:1–8
potentially ‘conjugative’ and ‘MOB’
plasmids.
Materials and Methods (2)

2 . A na l ys is o f A MR ge n e s i n s ma l l p l a s mi d s

• S ma l l p l asmid s ( Co l E 1 , Co l E 1 0 and I nc Q )  id e n tif ie d ba s e d o n p l as mid re p l ico n

s e q u e nce s u sing th e B L A STn a l go r ith m to l o o k fo r D N A h o mo l o g ie s in th e
G e n B ank d atabase .

• A M R g e n e s in MC P s  id e n ti fie d b y co mm an d - l i ne ve rs i o n o f A MR f i nd e r to o l ,
a nd Re s Find e r to o l  e nu me ratio n and c l as s if icati o n
Materials and Methods (3)

3. Bacteria, strains and culture conditions

• Cloning the poly(A) tail was used only for pCR2.1 armA.

• To c l o n e a r m A i n t o p A C Y C 1 8 4  a m p l i f i e d w i t h a r m A 4 0 0 a n d a r m A R
then digested EcoRI and cloned into the EcoRI site of pACYC184.

• Re ce p i e n t : E s c h e r i c h i a co l i st ra i n W G 5 l y s o ge n i c fo r Stx p h a ge s 9 3 3 W,
312 or 557,25 or a Cdt phage and E. coli strain DH5a lysogenic for Stx
p h a ge s 9 3 3 W, 3 1 2 o r 5 5 7 .

• WG5 is a nalidixic acid-resistant mutant of E. coli.

• D H5a is a re cA - n egat ive E . coli K- 12 d erivat ive.

• Stx p h a ge s 9 3 3 W, 3 1 2 a n d 5 5 7  Po d o v i r i d a e t h at ca r r y s h i ga tox i n .

• Cdt phage  Myoviridae that carr y the cytolethal distending toxin.

• All lysogens were transformed with MCPs (pACYC184, pCR2.1 or pUC18)

and low-copy plasmids (IncM2, IncFII or IncR).
Materials and Methods (4)
• 5 . P L A S M I D C O P Y N U M B E R Q U A N T I F I C AT I O N A N D
4. COMPLETE SEQUENCE OF PLASMIDS WITH ILLUMINA AND MINION
TECHNOLOGY
• D N A extractio n and p u r if icatio n 
W izard G e no mic D N A P u r i f i cati o n K i t .
• D N A q u al ity and co nce n tratio n we re
meas u re d by Nano D ro p.
• S e q u e nce s p ro ce ss e d s u bs e q u e nt
a nal y sis  ch e ck th e q u a l ity w i th
Fa stQ C ve rsio n 0. 1 1 . 3 and tr i mmi ng
e n d nu cl e otid e s o f l ow q u a l ity u s e
Tr immo matic ve rsi o n 0 . 3 3 .
QPCR FOR ARMA
• DNA extracted from 2ml LB broth and
quantified using Qubit
• Gene uidA was amplified to compare plasmid
ratio to chromosomal DNA
• C a l c u l a t e t h e e ff i c a c y o f r e a c t i o n u s i n g q P C R
with 5 dilutions DNA template
• Calculate plasmid copy number  cn = (1!
E c ) Ct c / ( 1 ! E p ) Ct p % S c / S p
• q P C R w i t h a n e ff i c i e n c y b e t w e e n 9 5 % a n d
100%  significant
• T h e a r m A q P C R a s s a y e ff i c i e n c i e s : 9 8 . 4 % ,
detection limit of 2.74 gene copies.
Material and Methods (5)
7 . P H A G E I N D U C T I O N A N D I S O L AT I O N O F P H A G E
6 . P H A G E I N D U C T I O N A N D I S O L AT I O N O F P H A G E PA R T I C L E S PA R T I C L E S

• Culture lysogen grown on LB broth  • Higher DNA concentration was required  m

incubated overnight in the presence of 1 0 0 m L c u l t u r e s o f s t r a i n s W G 5 - 9 3 3 W, W G 5 -
mitomycin C at 37C in the dark. 312 and DH5a-557.

• T h e Pe a r s o n c o r r e l a t i o n c o e ff i c i e n t ( r ) w a s • 50 mikroL of DNA that was digested with S1

c a l c u l a t e d b y t w o -t a i l e d c o r r e l a t i o n . P ≤ nuclease  transferred to nylon N+
0.05. membranes, analysed separatedly on 0.7%
a g a r o s e g e l s i n 0 . 5 - f o l d c o n c e n t r a t e d Tr i s
b o r a t e E D TA b u ff e r a n d s t a i n e d w i t h e t h i d i u m
bromide.

• The presence of armA and the plasmids was

determined in all the digested phage DNA by
S o u t h e r n b l o t h y b r i d i za t i o n .
Results and Discussion (1)

1. P l a s m i d s i z e s a n d m o b i l i za t i o n g e n e s

• 4 4 . 2 % o f t h e l a r g e p l a s m i d s p o s s e s s a f u l l T 4 SS .

• A n ot h e r s e t o f p l a s m i d s c a r r y o n l y a r e l a xa s e t h at e n a b l e s co n j u ga t i o n u s i n g t h e T 4 SS o f
a co - r e s i d e n t co n j u ga t i v e p l a s m i d .

• MOB plasmids 40.5% of small plasmids.

• S m a l l p l a s m i d s d o n ot n e e d t h e M O B g e n e s to b e e ff i c i e n t l y m o b i l i ze d t h r o u g h
co n j u ga t i o n .

• N o n - M O B p l a s m i d s w i t h a n o r i g i n o f t ra n s fe r ( o r i T ) ca n b e co n j u gate d b y r e l a xa s e s
e n co d e d i n co r e s i d e n t p l a s m i d s a c t i n g i n t ra n s .
2. Small plasmids confer
resistance to most antimicrobial
classes

• M C P s ( p CC K 6 4 7 4 1 o r
pMCR_R3445,42) carry a single
resistance gene, other resistance
genes were aligned in tandem (like
in pB1005)  would lead resistance
to se veral antimicrobial class
3. High-efficiency packaging of DNA from MCPs in phages

• Encapsidation rate of armA higher

when the genetic platform of armA
was an MCP than when it was carried
on a large low-copy plasmid (P <
0.0001).

• MCPs cannot transfer horizontally

(conjugation).

• Phages in our study use a cos-

packaging mechanism, which
eliminates the possibility of
generalized transduction.

• chromosomal lateral transduction 

how phage packaging can mediate
e ff i c i e n t t r a n s d u c t i o n o f c o m p l e t e
genomes

• lateral transduction  prophages

packaging DNA through the cos
mechanism can mobilize large
fragments of chromosome. no
mobilization of plasmids
• A D H 5 a h o st re cA negati ve  s i mil a r to th o s e o bta in e d w i th th e re c A - p o s i tive
stra in WG 5.

• At a l owe r rate , l arge p l a s mi d s co u l d be trans fe r re d betwe e n ba cte r ia th ro u g h

p h a ge s u p to 4 1% o f a l l l arge p l a s mi d s an al y s e d h e re a l s o l a ck mo bi l i zati o n
ge ne s .

• MCP transd u ctio n co u l d be an extre me l y e ff ic i e nt mea ns o f mo bi l izati o n o f A M R

ge ne s