Los días 7 y 8 de mayo organizamos en la Fundación Ramón Areces con la Fundación General CSIC el Simposio Internacional 'Microbiología: transmisión'. La "transmisión" en microbiología hace referencia al proceso por el que material genético es transferido de una célula a otra, de una población a otra. Es un proceso clave para entender el origen y la evolución de los seres vivos. El objetivo de esta reunión era conocer mejor la logística de la transmisión para ser capaces de modular o suprimir algunos procesos de transmisión dañinos.

1. The Team all over the years

2. Ecology and evolution of chromosomal gene transfer between environmental
microorganisms and pathogens: An exaptation tale

3. Horizontal gene transfer is neither a mechanism allowing canonical
step-wise evolution nor sex
Genetic events underlying evolution.
Mutation (including genome rearrangements): Allows stepwise
evolution by producing incremental changes in gene sequences.
Sexual recombination: Allows stepwise evolution by shuffling the
combination of alleles already present in the species
Horizontal gene transfer: Allows the acquisition of a novel trait,
not necessarily present before in the species, as a whole; evolution in
quantum leaps.

4. Martínez, J. L. (2008) Science 321: 365-367.
To pathogenic microparasites (viruses,
bacteria, protozoa, or fungi), we and other
mammals (living organisms at large) are
little more than soft, thin-walled flasks of
culture media.
Levin, B.R. & Antia, R. (2001) Science
292: 1112-1115
Human body is en extreme environmental, which is rich in nutrients

5. In the treated patient, bacteria require
being simultaneously virulent and
resistant to antibiotics
Virulence has evolved along
thousands to hundreds of millions of
years.
Acquired antibiotic resistance is
the product of decades of evoution
The use of antibiotics makes human body an
even more extreme environment
patOP
Picasso
Goya
J.L.Martínez,F.Baquero(2002)ClinicalMicrobiology
Reviews.15:647
Are there common rules in the evolution
towards virulence and resistance?

6. Intrinsic and acquired mechanisms of virulence and resistance
Opportunistic pathogens with an environmental origin can present several
virulence determinants, not acquired by horizontal gene transfer, as well as
antibiotic resistance genes.

7. Origin and dissemination of antibiotic
resistance genes
patOP
Similar situation applies for the
acquisition of virulence
determinants, although the
amplification cycle is less
frequent.

8. Virulence involves the acquisition of sets of
genes allowing adaptation to the new
habitat.
This may produce de-adaptation from the
previous habitat and speciation

9. Origin and dissemination of antibiotic
resistance genes
patOP
Single genes can provide
antibiotic resistance to organisms
that are already adapted for
growing in the human host.
Resistance is not linked to
speciation

10. Common virulence factors
Endotoxin (Lipid A of LPS)
Capsule
Colonization factors (attachment to surfaces)
Production of extracellular matrix that enhances
biofilm formation
Acquisition of growth factors (e.g. Fe)
Antigenic phase variation
Resistance to serum killing
Type III secretion
Toxins
Virulence factors as regular elements of the bacterial physiology
Bacterial structure
Bacterial metabolism
Bacterial defense from host
Bacterial injury to the host

11. Role of virulence factors in natural
ecosystems
patOP
Biofilm for attaching to surfaces, for cooperating and
for avoiding protozoan grazing
Tetrahymena after grazing Listeria. Several,
at the vacuoles have been digested, some
are still intact
P. aeruginosa susceptible to grazing
P. aeruginosa resistant to grazing
Matz,J.,Deines,P.,Jürgens,K.(2002)FEMSMicrobiol.Ecol.(2002)57-65
Biofilm formation is important in chronic infections of surfaces
Alginate overproduction avoids macrophages’ action over P. aeruginosa

12. Virulence determinants can be relevant
for surviving outside the host
patOP
Citotoxicity, is as relevant virulence determinant that can have
evolved to avoid nematodes’ and protozoans’ killing:
Prey/predator relationship in an unicellular world
K aerogenes PAO1
Navas, Cobas, G., López, JA,Talavera, M., Ayala, JA., Martínez, JL.(2007) Proc Natl Acad Sci USA.
104:13728-13731
Non-cytotoxic PAO1 mutant

13. Known functions of antibiotic resistance genes
patOP
Antibiotic inactivating enzymes
Macing,D.R.,Rather,P.N.(1999)FrontBiosci4:132-140
Udou,T.,Mizuguchi,Y.,Wallace,R.J.,Jr.(1989)FEMSMicrobiol.Lett.48:227-230

14. Resistance to antibiotics: Fighting back
Proc. Nat. Acad. Sci. USA Vol. 70 pp. 2276-2280,1973 Aminoglycoside Antibiotic-Inactivating Enzymes
in Actinomycetes Similar to Those Present in Clinical Isolates of Antibiotic-Resistant Bacteria
RAOUL BENVENISTE AND JULIAN DAVIES
The metabolic role of the aminoglycoside-modifying enzymes in actinomycetes is not known. Miller
and Walker have postulated that phosphorylated streptomycin might be important as a metabolic
precursor of streptomycin or to detoxify the antibiotic (7). The enzymes might also be required for
transport of these antibiotics in or out of the cell. Alternatively, they may have nothing to do with
antibiotic biosynthesis and may play a role in another biosynthetic process.
Nothing is known about the origin of R factors. The Watanabe hypothesis (4) provides a simple
molecular mechanism for their origin, but we can only speculate on the environmental and evolutionary
factors that play a role in their formation and maintenance. Their presence does not seem to require
the extensive use of a selective antibiotic environment since Gardner et al. (21) have found R factors in
an "antibiotic virgin population" in the Solomon Islands.

15. Known functions of antibiotic resistance
genes
Chromosomally-encoded aminoglycosides N-acetyltransferases (AAC)
Aminoglycoside-inactivating enzymes are classical antibiotic resistance
genes acquired through horizontal gene transfer.
However, several bacterial species that do not produce antibiotics
contain in their chromosomes genes encoding AACs that have not
recently acquired through horizontal gene transfer.
Examples: Providencia stuartii, Mycobacterium fortuitum,
Mycobacterium tuberculosis, Serratia marcescens, Acinetobacter
haemolyticus, Enterococccus faecium, Stenotrophomonas maltophilia,…
At least in the case of P. stuartii, the primary function of the
acetyltransferase is the modification of the peptidoglycan.
Macing,D.R.,Rather,P.N.(1999)FrontBiosci4:132-140
Udou,T.,Mizuguchi,Y.,Wallace,R.J.,Jr.(1989)FEMSMicrobiol.Lett.48:227-230

16. Known functions of antibiotic resistance genes
patOP
Multidrug efflux pumps Enzymes involved in bacterial
physiology and capable to
recognize the antibiotic

17. MDR pumps differ to classical antibiotic
resistance genes
Ubiquitously found in all
organisms
Redundandt (several efflux
pumps in a single organism)
Conserved structure
Conserved chromosomal
genes
Wide range of substrates
This suggests that MDR pumps are ancient
elements with relevant roles for bacterial
physiology
patOP

18. Expression of MDR efflux pumps
is usually repressed by local
regulators
SmeT
smeT smeD smeE smeF

19. smeT smeD smeE smeF
Overexpression
Expression of MDR efflux pumps is high in the presence of
effectors. Knowing the effectors provide clues on the
function of MDR pumps

20. SmeT does not bind antibiotics but binds plants
exudates
 EMSA
 Model
Specific binding SmeT-Inter smeT-D
DNA + Prot
DNA
SmeT functional mechanism

21. Media CFUs/g
0,00E+00
5,00E+08
1,00E+09
1,50E+09
2,00E+09
2,50E+09
3,00E+09
3,50E+09
D457 MBS411 D457R MBS82 GGL199 MBS108
Media
CFUs/g
** **
The deletion of smeE impairs the capability
of S. maltophilia for colonizing plants. Wild-type
smeE-deletion mutant
SmeDEF, which is the most relevant element
contributing to quinolone resistance in S.
maltophilia is involved in the endophytic
colonization of plants’ roots.
García-León, G., Hernández, A., Hernando-Amado, S., Alavi, P., Berg, G., Martinez, J. L. (2014). Applied and
Environmental Microbiology 80: 4559-4565

22. Exaptation as a major force in the evolution
of antibiotic resistance
AR genes acquired by HGT are expressed in
a different genetic context and in the
absence of their specific regulatory
networks. Their solely function is thus
antibiotic resistance
Martínez, J.L. (2009) Philosophical Transactions of the Royal Society B:
Biological Sciences 267: 2521-2530.

23. Resistance (and likely virulence) is
everywhere

24. Bottlenecks in acquisition
of genes from the
chromosomes of
environmental bacteria

25. Ecological connectivity

26. Antibiotic Use
Human
Microbiota
Animal
Microbiot
a
Hospitals
LTCFs
Farms
Aquaculture
Wastewater,
effluents
Sewage
treatment
plants
Soil,
Sediments
Surface,
ground
water
r r
r
r
r
r
r
r
r
r
r
r
r
r
r
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r
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r
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r
r
R
R
r
R
RR
R
R
The reactors for the building up of antibiotic
resistance
Baquero F, Martínez, J. L., Cantón R. (2008). Current Opinion in Biotechnology
19:260-5

27. Founder effect in the establishment of
antibiotic resistance genes
TEM-1 beta-lactamase is the first antibiotic resistance gene described.
Altought several derivatives of TEM-1 (and other plasmid-encoded
betalactamases) are selected in pathogenic bacteria, the number of completely
different plasmidic beta-lactamases is rather low considering the amount of
potential beta-lactamases in environmental bacteria.
T
P
Q
A
XW
Z
L
D P
P
T
P
Q
A
XW
Z
L
D P
P
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L
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P
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γ
φ
1 2 3 4
Martínez, J.L., Fajardo, A., Garmendia, L., Hernández, A., Linares, J. F., Martínez-Solano, L., Sánchez,
M. B. (2009) FEMS Microbiology Reviews 33: 44-65
This may also happen for virulence factors: once a T3SS has been
acquired another one would not be needed

28. The directed network of recent lateral gene transfers.
Ovidiu Popa et al. Genome Res. 2011;21:599-609
Copyright © 2011 by Cold Spring Harbor Laboratory Press
In the case of virulence, the elements
to be transferred must genetically
clustered in the potential donor

29. 1
23
45
6
a
b
c
d
e
f
a
b
Fitness costs associated to antibiotic
resistance
Antibiotic resistance may produce a burden on
bacterial metabolism because:
- Target proteins are very important for bacterial
physiology, so that antibiotic resistance mutations in
this proteins make them less proficient.
-The acquisition of a antibiotic resistance plasmid
renders a metabolic cost (replication, transcription,
translation).
However:
-Some resistance mutations produce no-cost
-Resistant mutants can acquire compensatory
mutations.
-The effect of resistance on bacterial competitivity is
dependant on the ecosystem.
-Systems of co-selection and plasmid stabilization allow
persistence of resistance.
-Metabolic rewiring can compensate fitness costs
Does it also apply for virulence ?

30. Marie Doyle et al. Science 2007;315:251-252
Differential fitness costs of virulence determinants

31. Published by AAAS
The paradoxical efect of H-NS on fitness costs:
Epigenetic effects and expression levels
Marie Doyle et al. Science 2007;315:251-252

32. Marie Doyle et al. Science 2007;315:251-252
High fitness costs of virulence determinants may imply speciation
Driver of speciation

34. Entering in a new host implies global changes in the physiology

35. Dongsheng Zhou, and Ruifu Yang Infect. Immun. 2009;77:2242-
2250
Entering in a new host implies global changes in the physiology: and
consequently in the regulatory networks

36. Evolution of bacterial pathogens: from natural ecosystems to
human hosts
Environmental Microbiology
Volume 15, Issue 2, pages 325-333, 1 AUG 2012 DOI: 10.1111/j.1462-2920.2012.02837.x
http://onlinelibrary.wiley.com/doi/10.1111/j.1462-2920.2012.02837.x/full#f2

37. Evolution of bacterial pathogens: from natural ecosystems to
human hosts
Environmental Microbiology
Volume 15, Issue 2, pages 325-333, 1 AUG 2012 DOI: 10.1111/j.1462-2920.2012.02837.x
http://onlinelibrary.wiley.com/doi/10.1111/j.1462-2920.2012.02837.x/full#f2
Differing to other processes of
recombination-driven evolution (sex), in
which the better adapted offspring
usually outcompete the ancestor. HGT
allows the colonization of a new habitat;
the ancestor can remain and is not
outcompeted by the evolved offspring.

38. patOP
Towards an integrated and ecological view
on bacterial virulence and resistance
Pathogens have acquired virulence traits and
resistance genes out of clinical settings.
Antibiotic resistance genes and virulence
determinants have a relevant ecological role out of
humans.
Infection is a way of gaining access to new niches
and food resources.
Virulence and resistance should have a
metabolic control.
Magritte

39. Marisa
Jorge
Agustín
Alvarina
Jaime
Fernando
Fernando
Víctor
¡Gracias!

41. Crc (Catabolic carbon repression): Global
regulator of resistance and virulence?
Virulence (and resistance) is under metabolic
control
Morales,G.,Linares,J.F.,Beloso,A.,Albar,J.P.Martínez,J.L.,Rojo,F.
(2004).JournalofBacteriology186:1337-1344
crcwt

42. Effect of Crc on the
virulence of P.
aeruginosa
Gene TIGR
Locus
Average
Ratio*
Protein name Main role Reported role in virulence or resistance Putative Crc-binding sequence
and position (relative to AUG)
hcp1 PA0085 -12.26 Conserved hypothe tical
protein
Type VI secreted effector Type VI secretion (Mougous et al., 2006)
tolB PA0972 -1.5 Transport of small molecules
PA1459 -2.39 Probable
methyltransferase
Chemotaxis and motility Likely swarming** (Yeung et al., 2009).
Susceptibility to several antibiotics
(Dotsch et al., 2009)
gltA PA1580 -1.78 Citrate synthase Energy metabolism Type III Secretion (Rietsch and
Mekalanos, 2006)
fabB PA1609 -1.71 Beta-ketoacyl-ACP
synthase I
Fatty acid and phospholipids
metabolism
tig PA1800 -1.59 Trigger factor Cell division, chaperones & heat
shock proteins
Resistance to quinolones (Breidenstein et
al., 2008)
hcnB PA2194 -1.52 Hydrogen cyani de
synthase HcnB
Central intermediary
metabolism. Biosynthe sis of
cyanide
Cyanide production (Ramette et al.,
2003). Susceptibility to several
antibiotics (Fajardo et al., 2008).
lpxA PA3644 -2.1 UDP-N-
acetylglucosamine
acyltransferase
Cell wall / LPS / capsule
fabZ PA3645 -1.78 (3R)-
Hydroxymyristoyl -[acyl
carrier protein]
dehydratase
Cell wall / LPS / capsule
pepA PA3831 -2.06 Leucine
aminopeptidase
Protein fate Cytotoxicity (Hauser et al., 1998).
Susceptibility to several antibiotics
(Dotsch et al., 2009). Alginate
(Woolwine and Wozn iak, 1999)
murC PA4411 -1.53 UDP-N-
acetylmuramate--
alanine ligase
Cell wall / LPS / capsule Peptidoglycan (Azzolina et al., 2001)
azu PA4922 -3 Azurin precursor Energy metabolism Cytotoxicity towards macrophages
(Yamada et al., 2002)
rho PA5239 -1.69 Termination factor Rho Transcription, RNA processing
and degradation
Swarming (Yeung et al., 2009)

43. Effect of Crc in the multicellular
behaviour of Pseudomonas
aeruginosa
The Crc mutant:
Is less motile
Forms large clumping structures
Produces more EPS
Attaches more efficiently to surfaces

45. Common virulence factors
Endotoxin (Lipid A of LPS)
Capsule
Antigenic phase variation
Acquisition of growth factors (e.g. Fe)
Resistance to serum killing
Antimicrobial resistance

46. patOP
Towards an integrated and ecological view
on bacterial virulence and resistance
Opportunistic pathogens have acquired virulence
traits and resistance genes out of clinical settings
(although they are acquiring novel antibiotic
resistance genes)
Antibiotic resistance genes and virulence
determinants have a relevant ecological role out of
humans.
Infection is a way of gaining access to new niches
and food resources.
Virulence and resistance should have a
metabolic control.
Magritte

47. Known functions of antibiotic resistance genes
besides resistance
patOP
Elements involved in
detoxification of metabolic
intermediates or
mediating bacterial
comunication.
Enzymes involved in
bacterial physiology and
capable to recognize the
antibiotic
Proteins that modulate
the activity of bacterial
targets

48. What is an antibiotic resistance gene?
A gene that contributes to the intrinsic resistance of a given
species
Its overproduction makes bacteria more resistance
Its absence makes bacteria more susceptible
Mutations in some genes as those coding targets, transporters
or regulators can confer resistance; but such genes are not
resistance genes because they will not confer resistance upon
transfer to a heterologous host. There are exceptions as the S.
pneumoniae topoisomerasesA gene that confers resistance upon its transfer to another wild-type bacterial
host
Given that the original function of several resistance genes is not antibiotic
resistance, inferring function by homology is risky
Technical definition

49. Martinez, J. L., F. Baquero & D. I. Andersson, (2007) Predicting antibiotic resistance. Nature Reviews in
Microbiology 5: 958-965.
Martinez, J. L., F. Baquero & D. I. Andersson, (2011) Beyond serial passages: new methods for predicting the
emergence of resistance to novel antibiotics. Curr Opin Pharmacol 11: 439-445.
Functional and sequence-based metagenomics
Functional analysis of comprehensive transposon-tagged
mutants
Molecular epidemiology
Whole-genome-sequence of
antibiotic resistant mutants
Conceptual framework for predicting acquisition of
antibiotic resistance by bacterial pathogens

50. Conceptual framework for predicting antibiotic
resistance
Martinez, J. L., F. Baquero & D. I. Andersson, (2007) Predicting antibiotic resistance. Nature Reviews in
Microbiology 5: 958-965.
Martinez, J. L., F. Baquero & D. I. Andersson, (2011) Beyond serial passages: new methods for predicting the
emergence of resistance to novel antibiotics. Curr Opin Pharmacol 11: 439-445.

51. Bottlenecks in the
acquisition of genes by
pathogens from natural
ecosystems

54. Acquisition of secondary mechanisms of
resistance may compensate fitness costs

55. Fitness costs can be gene-specific

56. Fitness costs in the presence of antibiotics: Antibiotic pollution favours
resistance even at non-inhibitory concentrations

58. In the treated patient, bacteria require
being simultaneously virulent and
resistant to antibiotics
To understand in full the infective
process in an antibiotic-overloaded
word, we need to understand the
networks connecting resistance and
virulence
patOP
Picasso
Goya
J.L.Martínez,F.Baquero(2002)ClinicalMicrobiology
If virulence is a way of gaining
access to the resources of a
given ecosystem, it has to be
under metabolic control

59. pBS3.25 coloniapBS3.25 (glicerol)pBS18
MC410
hns
30 horas
pBS18 (pGEM-T with qnrA1 gene)
pBS3.25 (pGEM-T with Smqnr25 gene)

60. Fitness cost of different qnr genes
Acquisition of Smqnr of S. maltophila has a fitness cost, which is not
observed in a ∆hns strain
KZM120 (∆acrAB) MC4100 JMG100 (∆hns)
0
20
40
60
80
100
120
pGEM-T
pBS3.25 pBS18 pBS19 pBS20
Cellswithplasmid(%)
Smqnr qnrA1 qnrA3 qnrA4
Escherichia coli

61. Mutations take place in the chromosome of Escherichia coli
Stability of pBS3.25 after evolution
No mutations in the plasmid
E. coli KZM120 pBS3.25
evol
0
20
40
60
80
100
120
pGEM-T MBS25 MBS228 MBS229 MBS230 MBS231
retr
Cellswithplasmid(%)

62. The paradoxical efect of H-NS on fitness costs

64. Source-sink model of evolution of bacterial
pathogens