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Experimental epidemiology of antibiotic
resistance: looking for an appropriate animal
model system
Amparo Latorre
Universi...
Outline
The problem
The goal
The model system: Blatella germanica
 Role of Blattabacterium
 Ecological succession of ...
Antibiotic resistance is widely recognized as one of the
major challenges in Public Health
Concept
The problem of transmis...
The problem
Hierarchy levels
Such multi-level complexity is difficult to address as a whole
We propose to establish an innovative, cheap, and
reproducible experimental model of transmission of
information about ant...
Two different environments
HospitalHospital
Individuals harboring bacterial
strains with AB resistance genes
Increased abu...
Why B. germanica…..a cockroach?
Is it possible to “humanize” cockroaches?
Blattella germanica: a model system
ENDOSYMBIONT...
B. germanica: a model system
 Five (males) or six (females) nymphal stages
 The entire cycle lasts about 100 days (from ...
Tissues:
 Fat body: Blattabacterium
 Gut: Foregut – Midgut – Hindgut
Microbial
communities
B. germanica: a model system
...
The role of Blattabacterium
Three cell types in the fat body
 Urate cells (U).
 Bacteriocytes (M).
 Adipocytes (L).
Uri...
López-Sánchez et al. 2009. PLoS Genetics
Patiño-Navarrete et al 2014. Biol Letters
The role of Blattabacterium
Predicted m...
adultegg n5n4n3n2n1
Ecological succession of the gut microbiota
Carrasco et al. 2014. Int. Microbiol
Perez-Cobas et al. 20...
Ecological succession of the gut microbiota
Scanning Electron Microscopy of the luminal surface of the hind gut of
B. germ...
To better understanding the system
To check for the essential role played by gut microbiota in host physiology.
To asses...
Work in progress
B. germanica treated with Antibiotics
Ootheca
hatching
Dissection 1
CD
adults
faeces
CD+AB
CD
n1
Ootheca
...
Identifying autochthonous cultivable bacterial strains
Gut extracted
Lab B. germanicaWild B. germanica
McConkey- Agar M-En...
Work in progress
Preliminary results
BHI (B)
Lab: none
Wild: 3 isolates
2 (Vanc+Kan)
Lab: 3 isolates
1 (Kan+Vanc+Rif)
1...
Two different environments
Hospital
Individuals harboring bacterial
strains with AB resistance genes
Increased abundance
i...
Proposed experimental design
Water
+AB1
Water
+AB2
AB1 Against aerobic bacteria
AB2 Against anaerobic bacteria
Horizontal ...
How to transform a roach natural population in a hospital-like population?
COCROACH NATURAL POPULATION: gut dissection
Met...
From experiment to simulation and back
Real animal model of experimental evolution of AB resistance
Parameters
Cockroach ...
Evolutionary Genetics group
Andrés Moya Amparo Latorre Puri Carrasco
Fernando Baquero Teresa Coque
Pablo Llop Marta Ibáñez...
¡Muchas gracias!
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Amparo Latorre - Simposio Microbiología: Transmisión

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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.

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Amparo Latorre - Simposio Microbiología: Transmisión

  1. 1. Experimental epidemiology of antibiotic resistance: looking for an appropriate animal model system Amparo Latorre Universidad de Valencia
  2. 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. 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. 4. The problem Hierarchy levels Such multi-level complexity is difficult to address as a whole
  5. 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. 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. 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. 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. 9. Tissues:  Fat body: Blattabacterium  Gut: Foregut – Midgut – Hindgut Microbial communities B. germanica: a model system Dissection
  10. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 23. ¡Muchas gracias!

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