Fernando de la Cruz presented a document on plasmid conjugation systems and controlling the dissemination of antibiotic resistance. The document contained the following key points: 1. The only element common among all conjugatively transmissible plasmids is the relaxase protein. Conjugative plasmids also contain VirB4 proteins. 2. Different types of plasmids - conjugative, mobilizable, and non-transmissible - were analyzed based on size. IncF and IncI plasmids frequently transfer antibiotic resistance. 3. COINs (conjugation inhibitory compounds) show potential to control the dissemination of multidrug resistance by inhibiting bacterial conjugation without affecting growth.

1. Fernando de la Cruz
Madrid, May 7, 2015
Genetic transmission from bacteria-to-bacteria
Comparative Genomics
(PLACNET)
Control of AbR dissemination
(COINs)

2. THE DIVERSITY OF PLASMID
CONJUGATION SYSTEMS
Introductory slides

3. Elements of bacterial conjugation
DONOR
RECIPIENT
Two corollaries:
1. The only element in common among
all conjugatively transmissible plasmids
is the relaxase
2. Conjugative plasmids have relaxases
and VirB4s (the most conserved protein
of T4SSs).

4. Distribution of conjugative, mobilizable and non-transmissible plasmids
(1,730 plasmid genomes) according to plasmid size
Smillie C, MP Garcillan-Barcia, MV Francia, EP Rocha and F de la Cruz.
2010. Mobility of plasmids. Microbiol Mol Biol Rev 74:434-52.

5. Main relaxase families in γ-proteobacteria
260 Tra+ plasmids
in NCBI γ-proteobacteria
(271 relaxases)
DPMT: 19 primer pairs
targeting 17 main subfamilies
theoretically detect 193/271 (71%) γ-
proteobacterial relaxases
Garcillan-Barcia et al. 2009. The diversity of
conjugative relaxases and its application in
plasmid classification. FEMS Microbiol Rev
33:657-87.
Alvarado et al. 2012. A Degenerate Primer
MOB Typing (DPMT) Method to Classify
Gamma-Proteobacterial Plasmids in Clinical
and Environmental Settings. PloS One 7,
e40438.

6. 1
2
1
Permissive host Non-permissive host
3
Relationship between transmissible plasmids and ICEs
A. There are twice as many ICEs than plasmids in sequenced genomes
B. ICEs and plasmids are indistinguishable by phylogeny or protein signatures
C. Plasmids and ICEs frequently interconvert (at an evolutionary scale)
D. A substantial fraction of integrated elements were domesticated as protein
secretion specialists
August 2011 | Vol 7 | e1002222

7. PLASMID ANALYSIS FROM WGS DATASETS
Plasmid Genomics

8. PLACNET = Plasmid Constellation Networks
PLACNET components
2 types of nodes
 Contigs
 Reference sequences
2 types of edges
 Scaffold links
 Homology to references
In PLACNET, a plasmid is a connected component (a constellation of contigs)
The network contains one or more disjoint connected components:
chromosomes and plasmids
Staphylococcus aureus 118 ST772
Chr: 2.9 Mb
Plasmid: 12,819 kb
Lanza et al. (2014). Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation
network (PLACNET), a new method for plasmid reconstruction from whole genome sequences. PLoS Genet
10: e1004766.

9. Example I. ESBL+
ST131 E. coli
3.629.034 bp core genome

10. IncF plasmids
(ESBL genes)
Hierarchical
clustering
dendrogram of
reconstructed
ST131 plasmids
43 plasmids
15 MOB families

11. 252 protein families

12. Example II. ESBL+ E. coli of same ST type isolated from
humans and animals (32 strains =148 plasmids)

13. Phylogeny of reconstructed MOBP12 plasmids demonstrates
plasmid transmission between animals and humans

14. Hierarchical clustering dendrogram of reconstructed
MOBP12/IncI plasmids

15. THE WORLD OF PLASMIDS
Plasmidomics

16. 148 plasmids
70 plasmids
37 plasmids
59 strains 255 plasmids (4.3 plasmids / genome)
Distribution of plasmids in three Escherichia coli collections
32 ESBL-positive
strains
Different locations in The Netherlands
2006-2011
Source: human, chicken, chicken meat, pig,
de Been et al. Plos Genet 10: e1004776
19 Carbapenem-R
strains
Bioproject (PRJNA202876)
Submitted by Broad Institute (May 2013)
Source: human clinical isolates (three
hospitals in Boston-USA)
de Toro et al. Mic Spectrum (in press)
8 ST131 strains
4 strains virotypes A, B, C and D (Blanco et al., 2013)
4 strains (Bioproject PRJNA186413)
Source: human, Lanza et al. Plos Genet 10:
e1004766

17. 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5 5.1 5.2 5.3 5.4 5.5
0
5
10
15
20
25
30
35
40
MOBH11
MOBC12
MOBC1
MOBF12
MOBF11
MOBQ12
MOBQu
MOBP12
MOBP11
MOBP6
MOBP5
MOBP3
MOBV2
No-MOB
Size of plasmids (Kb)
[logarithmic scale]
Totalnumber
Distribution of MOB types (255 E.coli plasmids)
1 1.5 3 5 10 30
100 300
Smillie C, Garcillán-Barcia MP, Francia MV, Rocha EPC, de la Cruz F.
Mobility of plasmids. Microbiol Mol Biol Rev 74, 434–52 (2010).
Garcillán-Barcia MP, Alvarado A, de la Cruz F. Identification of bacterial
plasmids based on mobility and plasmid population biology. FEMS
Microbiol Rev 33, 936-56 (2011).
De Toro M, Garcillán-Barcia MP, de la Cruz F. (2014) Microb Spectrum (in
press)

18. 3 3.1 3.23.3 3.43.5 3.6 3.7 3.83.9 4 4.1 4.24.3 4.4 4.5 4.6 4.7 4.84.9 5 5.1 5.25.35.4 5.5
0
5
10
15
20
25
30
35
40
Rep-3 [pfam01051]
Rep-HTH-Crp2 [pfam13545]
Rep-1 [pfam01446]
Rep-HTH-27 [pfam13463]
Rep-HTH-36 [pfam13730]
RepFIB (pILF82-like)
RepQ12 (pSC101-like)
RepQu (pMG828-2-like)
RepQu (pIGWZ12-like)
RepQu (ColE9-J-like)
IncHI2
IncHI1
IncI2
IncI1, IncK, IncB/O
IncN
IncX
IncP1
IncF
ColE1-like
N.D.
Size of plasmids (Kb)
[logarithmic scale]
Totalnumber
Distribution of Inc-types (255 E.coli plasmids)
1 1.5 3 5 10 30
100 300

19. Some pending questions
(for plasmid biology)
 Why some plasmids appear frequently and others do not?
 What is unique about IncF or IncI plasmids and their
adaptation to E. coli?
 What are the causes for synergistic plasmid combinations
(e.g., IncF and ColE)? TRA/MOB?
 Why so many cryptic (selfish?) no-MOB plasmids?

20. CONTROL OF ABR
DISSEMINATION
COINs

21. Disarming MDR/Vir bacteria
Of the 3 potential target processes, only conjugation
seems to occur by a single major mechanism
Lesson from bacteria: Fight AbR dissemination now, or it might be too late!

22. Finding COINs:
R388-based high throughput conjugation assay
Fernandez-Lopez et al. (2005) Unsaturated fatty acids are inhibitors of bacterial
conjugation. Microbiol 151:3517-26.

23. COINs specificity

24. COINs act on donor cells and
are effective in all tested hosts

25. Halting plasmid dissemination with COINs
Effect of 2-HDA on the kinetics of plasmid
R1drd19 conjugation in liquid medium
COINs effectively inhibit
plasmid infection of a
recipient population
Experiments should be
reproduced in natural
environments
We are testing a
freshwater microcosms
for inhibition of
MOBF12/IncF plasmid
infection by uFAs

26. MGEs and timescales in bacterial evolution
1. Short scale (few years): Antibiotic resistance
(the first evolution experiment at a planetary scale)
• Diversity
• Novelty
• Globality
... Appearance of MGEs (plasmids and Tns)
2. Intermediate scale (hundreds of years): Degradation of
xenobiotic compounds
(more local, more complex)
same mechanisms but:
• Sometimes phages
• Sometimes inserted in chromosomes
3. Large scale (millions of years): Pathogenicity
(slower, even more complex)
• Mostly genomic (pathogenicity) islands
““Horizontal gene transfer and the origin of species: lessons from bacteria”Horizontal gene transfer and the origin of species: lessons from bacteria”
de la Cruz and Davies (2000)de la Cruz and Davies (2000) Trends in Microbiol.Trends in Microbiol. 88: 128–133.: 128–133.

27. Acknowledgements
The UC team
Other collaborators
Julien Guglielmini, Eduardo Rocha (Institut Pasteur, Paris)
Marta Martinez, Antonio Fernandez (Instituto Biomar, León)
Teresa Coque, Val F. Lanza, Fernando Baquero (Hosp RyC, Madrid)
Jorge Blanco (Univ. Santiago de Compostela)
Mark de Been, Willem van Schaik, Rob Willems (Univ. Utrech)