El 29 de marzo de 2016 celebramos un Simposio Internacional sobre el 'Impacto de las ciencias ómicas en la medicina, nutrición y biotecnología'. Organizado por la Fundación Ramón Areces en colaboración con la Real Academia Nacional de Medicina y BioEuroLatina, abordó cómo un mejor conocimiento del genoma humano está permitiendo notables avances hacia una medicina de precisión.

1. Antibiotic Resistance Genes:
The Intestinal Resistome
Fernando Baquero
Department of Microbiology
Ramón y Cajal University Hospital
IRYCIS, CIBERESP
Madrid, Spain

2. THE GENOCENTRIC REVOLUTION
NOT the organisms,
but
the GENES,
as units for understanding
Nature

3. The ensemble of antibiotic resistance genes hosted in
all bacterial populations (culturable and non-culturable)
of
microbial communities in particular environments, as
intestinal, sewage, soil microbiomes…
The Resistome
Challenges for the Screening and Analysis of Resistomes
• Current definition of antibiotic resistance genes
• Noise in antibiotic resistance databases
• Limitations of High-Throughput Technologies
• Underestimation of size and diversity of resistomes

4. 4Nature MR Feb, Jun 2015

5. Antibiotic Resistance Genes
• In a broad sense, genes involved in the insusceptibility of
microorganisms to antimicrobial compounds
By random transposon-tagged
mutagenesis, at least 2 % of all
P. aeruginosa genes have
resistance effects:
the intrinsic resistome
Green, more antibiotic susceptible;
Red, more antibiotic resistant
Fajardo, A., .. Baquero, F.& Martínez, J. L. (2008). The neglected
intrinsic resistome of bacterial pathogens. PloS one, 3(2)

6. Plasmid R genes
Chromosomal
R genes
Antibiotic Resistance Genes
Genes encoding
specific mechanisms

7. Antibiotic Resistance and Antibiotic Resilience
in the Intestinal Resistome
• Resilience genes in MAJORITY bacterial populations:
Mainly chromosomal genes, mostly with house-keeping
functions (intrinsic resistome) assuring the healthy
maintenance of the (generally majority) host bacteria in their
environment, when antibiotics are present. Healthy bacterial
populations are maintained, but non selected by antibiotic
exposure.
• Resistance genes in the MINORITY bacterial populations:
Genes with a specific function on antibiotic resistance, frequently
to clinically used antibiotics, frequently associated to mobile
genetic elements (extrinsic, acquired resistome) spreading
among to minority-dangerous bacterial populations, which are
selected by antibiotic exposure. Only minorities are selectable!

8. Antibiotic Resistance Genes
• NOT necessarily genes listed in Antibiotic Resistance Databases
Frequent Mistakes in Databases
by including:

9. JL Martínez, T Coque, F Baquero (2015)
Nature Rev. Microbiol., 13:116-123
Ranking the Risks of
Detection of Resistance
Genes in Resistomes
Mobile Genetic Elements as
Vehicles of Antibiotic Resistance
Genes
Known genes
inactivating
antibiotics in-use
Location in human pathogens
(frequently minorities)
Start here

10. Vertical descent
Horizontal descent
Plasmids spread resistance genes from cell to cell
Spread of antibiotic resistance genes

11. The reasons for implementing
epidemiological surveillance of
genes involved in antibiotic
resistance

12. Deaths as a direct result of antibiotic resistance
“At least…”
• 25,000 deaths/year because Ab-R in Europe (EMA-EUROPA, 2012)
• 23,000 deaths/year (CDC Ab-Resistance Threats in the US, 2013)
• Immuno-depressed patients
• Elderly (>75)
• Admission to the ICU
• APACHE II score at ICU admission
• Large spectrum antibiotics
• Special treatments
• Rhinogastric tube in place
• Nutritional immuno-deficiency
• Poor medical care, shortage ICUs
• Resistance to cheap available agents
Developing countries:

13. • The incidence of nosocomial bacteremia due to highly-resistant microorganisms increased
8.7 times as fast as non-resistant ones (1996-2005)
• Moreover, the increase in resistance does not replace infections with susceptible pathogens,
but adds to the total burden of bacteremia
• Hospitalized patients have become progressively more severely ill over the last 10 years.
(Ammerlaan HSM, Bonten MJM et al., JAC 63:1064–1070, 2009)
Tedim ASP et al., AEM 2015
Enterococcus faecium
E. coli phylogroup B2
Irene Rodriguez ECCMID 2014
Bacteremia
RYC Hospital (1996-2012)
Antibiotic-R increases the number of severe infections
resistant

14. • Increase in total local number of resistant strains means increase the
absolute amount of resistance genes, and mobile genetic elements
gene
integron
transposon
plasmid, ICE
Antibiotic resistance
might increase the
density of tools involved
in microbial genetic
interactions,
with
unpredictable
consequences
Antibiotic resistance accelerates microbial evolution

15. Selection of incoming clone +
selection of the resident homologous clones
High-speed “global world dissemination” of R-clones
Example of global dissemination: Petty NK et al., Proc Natl Acad Sci U S A. 2014, 111(15):5694-9 (E. coli ST131)

16. Antibiotic release and antibiotic resistance altering
the environmental life-sustaining microbiosphere
• Possible ecological functional disturbances by the antibiotics in the environment include nitrogen
transformation, methanogenesis and sulfate reduction
• For instance, Cyanobacteria, largely susceptible to antimicrobial agents, as such type of organisms
accounts for more than 70% of the total phytoplankton mass, and are responsible for more than a third of the
total free O2 production, or CO2 fixation.
(Baquero F et al., Curr. Op. Biotechnol. 2008; 19:260–265; Dias E, et al. Frontiers in Microbiology 2015)
• Naturally-antibiotic resistant organisms might substitute under extended antibiotic pollution critical
susceptible organisms of huge ecological interest. Alternatively, acquisition of antibiotic resistance might
alter fitness or critical functional traits.
• Antibiotic disturbance of interbacterial signaling (semiotic space) with microecological consequences

17. Antibiotic-R altering the human-microbiota interactions
Human-microbiota common evolutionary history
Bacteroidetes
Clostridium
C-I
Clostridium
C- XIVa
Proteobacteria
(Enterobacteriaceae)
• Most of the significant antibiotic-resistance genes are located in minority
bacterial populations organisms
• When minority bacterial populations reach majority, there is an ecological
disturbance that might be deleterious for the system –unpredictable
clinical consequences under global overuse of antibiotics
• Consequences of acquisition of resistance by members of majorities?
M. Rajilic´-Stojanovic et al.
EM. 2012

18. Surveillance of Antibiotics Resistance Genes:
The Needs
• Significant antibiotic resistance genes: Focus those genes that influence
the outcome of infections treated with antimicrobial agents
• Significant allelic variants of antibiotic resistance genes: Allelic gene
variants might produce different resistance phenotypes.
• Genes involved in antisepsis and/or co-selection of antibiotic resistance:
resistance to biocides, heavy metals
• Genes identifying antibiotic resistance gene-spreading vectors: mostly
plasmids, able to disseminate genes in bacterial populations by horizontal gene
transfer.
• Genes present in minority populations in the microbiomes!!
18
D'Costa et al. Science 2006; Wright et al, NMR 2007; Wright GD, Expert Opin Drug Discov , 2010; Martinez, Coque and
Baquero, NMR 2015

19. Draussen buntes Leben (Paul Klee, 1931)
Colourful life outside
Enterococcus
Escherichia coli
Colourful intestinal microbiota Infections of intestinal origin
The bacterial minorities

20. ResCap: A targeted metagenomics approach
Targeted Metagenomics to Track and Characterize the Antimicrobial Resistome (ResCap1.0) Val F.
Lanza, Fernando Baquero, José Luís Martínez, Ricardo Ramos, Bruno González-Zorn, Antonio
Sánchez-Valenzuela, Fernando de la Cruz, and Teresa M. Coque (submitted 2016)
In Sequence-based metagenomic approaches (“open formats” as Metagenomic
Shotgun Sequencing MSS) antibiotic resistance genes from bacterial taxons that are
abundant in the population will be predominantly detected.
Arrays (“closed formats”) will be less affected by the more abundant populations.
The specificity of gene detection in both open and closed formats is greatly influenced by
detection of partial sequences that preclude a comprehensive understanding of diversity
ResCap WORKFLOW
i) Whole-metagenome shotgun library construction
ii) Hybridization with sequences of targeted Ab-R genes, including variant sequences
iii) Capture.

21. Draussen buntes Leben (Paul Klee, 1931)
Colourful life outside
Enterococcus
Escherichia coli
Colourful intestinal microbiota Infections of intestinal origin
The bacterial relevant minorities
E. coli genome: 0,.1 % of metagenome
E. coli AbR gene: ≈ 0.02% E. coli genome
Currently ResCap assures about 50 reads/AbR gene
in E. coli

22. HMM Profiles
Non-redundant, well identified
antibiotic resistance genes
UniRef100
Manual
Curation
Capture Platform
(ResCap)
≈81.000 nr genes
Relaxases
(plasmid markers)
Metal &
Biocide- R
EMBL CDS
Resistance-Capture
Platform Design
47,806
30,794
2,517
7,963

23. Based on SeqCap EZ, a single-step solution-based
capture method for enrichment of targeted
sequences in a single test tube

24. Comparison of Res-Cap
with conventional
Metagenomic Shotgun
Sequencing (MSS)
Sampling
Bacterial particles
Lysis, DNA extraction
Cloning, library construction
Sequencing clones
Sequence assembly into contigs and scaffolds
Comparison of MSS data
compared with ResCap
targeted sequence capture data
in fecal samples from 10
humans and 8 swine.

25. ResCap Platform
Efficiency
Number of detected known genes
per million of sequenced reads
Number of mapped reads per
million of sequenced reads

26. Abundance: read per Kilobase /per
million of reads that mapping against
genes/allele-clustered genes of
each AB-R family.
Diversity (number of DGCs detected per
sample): number of detected Genes Per
Million reads of each AB-R family.
Comparison of ResCap
and Conventional
Sequencing protocol in
Resistome Analysis

27. Reads/kb (normalized per gene size)
ResCap and Conventional Metagenomics in the detection of Antibiotic Resistance Genes

28. Glycopeptides
vanA
ResCap and Conventional Metagenomics in the detection of Antibiotic Resistance Genes

29. ResCap versus conventional metagenomics: abundance and
diversity of biocide-resistance putative genes

30. ResCap versus conventional metagenomics: abundance and
diversity of relaxase genes (plasmid type markers)

31. ResCap and Intestinal Resistome
• The ResCap v0.1 substantially enhances the sensitivity and
specificity of metagenomics shotgun sequencing (MSS) for
detecting and characterizing the resistome.
• Moreover, the ability to accurately detect genes from low
abundant or rare populations and its robustness, made ResCap
a promising tool for a wide number of research and diagnostic
applications and suitable for being used in both longitudinal and
cross-sectional studies.
• The platform constitutes one of the first examples of targeting
metagenomics for the analysis of bacterial populations.

32. Antonio S. Valenzuela
Val F. Lanza
Teresa Coque
me
Bruno
Gonzalez-Zorn
Fernando
de la Cruz
(Cantabria
University)
(UCM)
Jose Luis
Martínez
(CNB, CSIC)
Ricardo Ramos (PCM)

34. Copper-R genes Zinc-R genes
Arsenic-R genes Mercury-R genes

35. Close Environment
Internalized environment
Host-
microbiota
interphase
Microbiota
Inside
Intestine as “invironement”

36. Sick Microbiota
influences:
• Body development: stature, height
• Obesity (fat storage, dyslipidemia), lean-body mass
• Food allergy, allergies
• Resistance to invasive infections, bacteremia, UTI, surgical infections
• Prevention of infections (immunity)
• Tolerance to starvation
• Tolerance to particular diets
• Malabsorption
• Pharmacokinetics of drugs
• Inflammatory bowel diseases, ulcerative colitis
• Metabolic syndrome, atherosclerosis
• Hypertension
• Neurodegenerative disorders
• Autoimmune diseases
• Type I and type II Diabetes, insulin-resistance
• Behavior (The mind-body-microbial continuum)
• Bacterial vaginosis, Candida infections
• Skin disorders
• Coagulation, fibrinolysis
• Necrotizing enterocolitis, diarrhea, C. difficile
• Colorectal carcinogenesis; other?
• Spread of dangerous bacterial clones
• Antibiotic resistance

37. 1 E. coli ≈ 5 Mb
1 AbR gene ≈ 1 kb
1 AbR gene ≈ 0.02% E. coli genome
50.000.000 reads/sample
1% ≈ 0.1% Proteobacteria
E. coli ≈ 0.1% Metagenome
50 reads/AbR gene in E. coli
Why a Capture Platform?
Enough for
Quantification and/or
Identification??
The math-metagenomic approach