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. Experimental epidemiology of antibiotic
resistance: looking for an appropriate animal
model system
Amparo Latorre
Universidad de Valencia

2. Outline
The problem
The goal
The model system: Blatella germanica
 Role of Blattabacterium
 Ecological succession of the gut microbiota
Work in progress
Proposed experimental design
From experiment to simulator and back

3. Antibiotic resistance is widely recognized as one of the
major challenges in Public Health
Concept
The problem of transmission of antibiotic resistance
should be considered as derived from a constant flow of
information across multi-hierarchical biological
interactions, involving subcellular (resistance genes
located in plasmids, transposons, integrons), cellular
(clones) and supracellular (clonal complexes, genetic
exchange communities, microbiotic ensembles) levels.
The problem

4. The problem
Hierarchy levels
Such multi-level complexity is difficult to address as a whole

5. We propose to establish an innovative, cheap, and
reproducible experimental model of transmission of
information about antibiotic resistance using an
experimental model system, for a better understanding of
the dynamics of AB resistance genes in human
populations.
The goal
Objectives
To look for natural antibiotic resistant bacteria
To characterize and localize the resistant markers (chromosome, plasmid,
transposons, integrons, etc)
To introduce selective pressure to increase the resistant bacteria
To analyze transmission across hierarchies and environments
And more…

6. Two different environments
HospitalHospital
Individuals harboring bacterial
strains with AB resistance genes
Increased abundance
in resistant bacteria
UrbanUrban
Less
exposed
to AB
Migration
The goal
Experimental epidemiology of Antibiotic Resistance
To simulate two populations placed in two compartments, one with frequent
antibiotic exposure (mimicking a hospital), and the other with minimal or none
antibiotic exposure (the community), with a certain rate of migration between the
environments.
Highly
exposed
to AB
Individuals harboring bacterial
strains with AB resistance genes

7. Why B. germanica…..a cockroach?
Is it possible to “humanize” cockroaches?
Blattella germanica: a model system
ENDOSYMBIONT
(Blattabacterium)
ECTOSYMBIONTS
(many gut bacteria))
A COMPLEX Symbiotic System
 Similar to human gut microbiota
 A reservoir of AB resistance genes
An obligate mutualistic bacteria

8. B. germanica: a model system
 Five (males) or six (females) nymphal stages
 The entire cycle lasts about 100 days (from egg to adult)
 The nymphal instars last about 36-40 days
 Adult life span about 300 days
N1
Ootheca N2
N3
N4
N5
Male
Female
Lab-reared individuals come from populations maintained in the laboratory for 30 years at 26 °C;
12 hours of light and 12 hours of darkness. 70% humidity. Fed dog food and water ad libitum
Adult
Nymph
Nymph
Color: Pale Brown
Size: 1,3 to 1,6 cm
Wings: Both sexes with wings. Rarely fly
Habitat/location: cracks and crevices of
walls, sewer systems, etc

9. Tissues:
 Fat body: Blattabacterium
 Gut: Foregut – Midgut – Hindgut
Microbial
communities
B. germanica: a model system
Dissection

10. The role of Blattabacterium
Three cell types in the fat body
 Urate cells (U).
 Bacteriocytes (M).
 Adipocytes (L).
Uric acid storageBlattabacterium
Obligate intracellular bacteria.
Aerobic, Gram negative
Blattabacterium cuenotii
- Phylum Bacteroidetes.
- Class Flavobacteria.
López-Sánchez et al. 2009. PLoS Genetics
Genome Features Blattabacterium Bge
Genome size (bp) 636,850
G+C content (%) 27.1
Total number of genes 627
CDSs 586
rRNAs 3
tRNAs 34
Other RNAs 3
Pseudogenes 1
Overall coding región
(%)
96.3
CDSs average length (bp) 1,034
Accession number CP001487

11. López-Sánchez et al. 2009. PLoS Genetics
Patiño-Navarrete et al 2014. Biol Letters
The role of Blattabacterium
Predicted metabolism
Synthesis of essential amino acids
Nitrogen metabolism
Proposed model involving host activities
Transcriptome sequencing and comparative
analysis of different B. germanica tissues

12. adultegg n5n4n3n2n1
Ecological succession of the gut microbiota
Carrasco et al. 2014. Int. Microbiol
Perez-Cobas et al. 2015. FEM Microbiol Ecol..
2 11 15 22 34 68
Days after hatching
Microbial diversity: 100 different
families distributed in 13 phyla
 Microbial composition differs between
adults and nymph
 Fusobacterium accumulates with age
while Bacteroides decreases
 Blattabacterium is the only bacterium
found in the ootheca
The bacterial load increases two order of
magnitude from n1 to n2, coinciding with the
incorporation of the majority of bacterial taxa
Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria

13. Ecological succession of the gut microbiota
Scanning Electron Microscopy of the luminal surface of the hind gut of
B. germanica
Rich and dense bacteria biofilm
Filamentous morphotype bacterium
A and C: 50µm
B and D: 5µm
C,D n3 instar nymph
A,B Adult
Rich and dense bacteria biofilm

14. To better understanding the system
To check for the essential role played by gut microbiota in host physiology.
To asses the effect of AB in the symbionts of B. germanica and in the host
fitness.
To assess the transmission way of gut microbiota.
 From faeces, environment, trophallaxis, dead animals.
 To prepare synthetic diets.
To the proposed project
 To look for natural culture antibiotic resistance bacterial strains in wild
and in lab-reared populations.
 To locate and characterize the resistance genes.
 Metagenomics of natural and lab-reared populations (to estimate the
frequency of the antibiotic resistance genes, bacterial
composition,etc).
 To design markers to follow transmission.
Work in progress

15. Work in progress
B. germanica treated with Antibiotics
Ootheca
hatching
Dissection 1
CD
adults
faeces
CD+AB
CD
n1
Ootheca
appearance
CD + Rif
CD + Faeces
CD
Dissection 2 Dissection 3
Rifampicin
Control
Faeces
Dissection
Fat body: qPCR to monitor Blattabacterium densities
Gut: 16S rRNA Illumina sequencing to monitor changes
in bacterial composition and Metagenomics in selected
samples
Fitness parameters of B. germanica
Weight, offspring, developmental time
Recovery?
Decreasing
Unchanged
Microbiota
composition
CD, control diet

16. Identifying autochthonous cultivable bacterial strains
Gut extracted
Lab B. germanicaWild B. germanica
McConkey- Agar M-Enterococcus-Agar
Plated on
Morphology diversity
Isolation and purification
Work in progress
BHI
Plating with AB
Species identification. Resistance genes involved

17. Work in progress
Preliminary results
BHI (B)
Lab: none
Wild: 3 isolates
2 (Vanc+Kan)
Lab: 3 isolates
1 (Kan+Vanc+Rif)
1 (Kan)
1 (Rif)
Wild: 9 isolates:
1( Kan+Vanc+Rif)
1 (Kan+Vanc)
3 (Vanc)
2 (Van+Rif)
1 (Rif)
McConkey- Agar (Mc)
Lab: 2 isolates
1 (Kan)
Wild: 1 isolate
1 (Vanc)
M-Enterococcus-Agar (E)
Strain Kanamycin
(50 µg/ml)
Vancomycin
(3 µg/ml)
Rifampicin
(50 µg/ml)
Gram Identification (16S rRNA gene)
BLab1 + + + - Pseudomonas geniculata/
Stenotrophomonas pavanii
BLab2 - + + Enterococcus durans
BLab3 + -
BW1 - + - Klebsiella oxytoca
BW2 - + - Klebsiella oxytoca
BW3 + + -
BW4 + + + - Pseudomonas nitroreducens
(multiresinivorans)
BW5 - + + - Pseudomonas nitroreducens
(multiresinivorans)
BW6 - + + +
BW7 - - +
BW8 - + - Pseudomonas nitroreducens
(multiresinivorans)
BW9 - + + Klebsiella oxytoca/
Citrobacter freundii
McLab1 - -
McLab2 + +
McW1 - + - Klebsiella oxytoca
EW1 + + -
EW2 + + -
EW3 - - +

18. Two different environments
Hospital
Individuals harboring bacterial
strains with AB resistance genes
Increased abundance
in resistant bacteria
Urban
Less
exposed
to AB
Migration
Experimental epidemiology of Antibiotic Resistance
Highly
exposed
to AB
Individuals harboring bacterial
strains with AB resistance genes

19. Proposed experimental design
Water
+AB1
Water
+AB2
AB1 Against aerobic bacteria
AB2 Against anaerobic bacteria
Horizontal transfer events of bacteria, plasmids, genes
Cockroach population
Cockroach
Bacteria
Plasmids
Resistance gene
Colour Code
Migration
HospitalHospital UrbanUrban
Transfer events
Cockroaches migration

20. How to transform a roach natural population in a hospital-like population?
COCROACH NATURAL POPULATION: gut dissection
Metagenomics analysis Plating on general/selective media
Isolation of bacteria Screening for ABR
Resistance genes/locations
Analyzing the resistome
Frequency estimation
NATURAL BACTERIA WITH RESISTANCE GENES AND WITH A REPORTER PLASMID
INTRODUCED IN THE NATURAL POPULATION
Hospital-like: bacteria with natural (known) resistance genes, and a reporter (GFP, other)
Plasmid isolation
Plasmid modificaction with a reporter gene
Proposed experimental design

21. From experiment to simulation and back
Real animal model of experimental evolution of AB resistance
Parameters
Cockroach numbers (avoiding overcrowding)
Bacterial composition (“metagenomics”)
Plasmids (“plating and isolation” and “metagenomics”)
AB resistance genes (plating and metagenomics)
AB supply (kinds and dosages)
Migration rates (several)
etc
A Membrane Computational model to investigate the transmission of AR across the Trans-
Hierarchical-levels
Marcelino Campos, Carlos Llorens, José María Sempere, Ricardo Futami, Irene Rodriguez, Purificación Carrasco,
Rafael Capilla, Amparo Latorre, Teresa M. Coque, Andrés Moya, Fernando Baquero*
Biology Direct (submitted)

22. Evolutionary Genetics group
Andrés Moya Amparo Latorre Puri Carrasco
Fernando Baquero Teresa Coque
Pablo Llop Marta IbáñezCarlos GarcíaJuli Peretó
Entomology and Pest Control
Ximo Baixeras

23. ¡Muchas gracias!