Xenobiotics and Xenogenetics: Are humans increasing bacterial evolvability? - Michael Gillings
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Xenobiotics and Xenogenetics: Are humans increasing bacterial evolvability? - Michael Gillings

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This presentation will discuss the spread of antibiotic and resistance determinants from human waste streams into natural environments, and the likely consequences for microbial evolution.

This presentation will discuss the spread of antibiotic and resistance determinants from human waste streams into natural environments, and the likely consequences for microbial evolution.

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Xenobiotics and Xenogenetics: Are humans increasing bacterial evolvability? - Michael Gillings Xenobiotics and Xenogenetics: Are humans increasing bacterial evolvability? - Michael Gillings Presentation Transcript

  • Michael Gillings Department of Biological Sciences and Genes to Geoscience Research Centre, Macquarie University michael.gillings@mq.edu.au
  • Humans are having measurable and dramatic effects on • Atmosphere • Hydrosphere • Biosphere 1. Sequestration of a large proportion of primary production 2. Extinction of a wide range of taxa 3. Pollution with bioactive compounds 4. Accelerating evolutionary change by selection It is clear that we have precipitated evolutionary changes by both artificial and natural selection, but are we also changing the basal rates at which evolution can occur? Palumbi 2001 Science 293: 1786-1790
  • Is arguably the outstanding example of evolution by natural selection Mutation and lateral transfer of genes between cells and species drives this phenomenon
  • Antibiotics select for mutations and lateral transfer events that confer resistance. Large quantities of antibiotic are excreted unchanged.
  • Waste streams release resistance determinants and their DNA vectors simultaneously with disinfectants, antibiotics and heavy metals. This creates a hotspot for complex interactions between DNA elements in an environment containing sub-inhibitory concentrations of diverse selective agents. Schluter et al. 2007 FEMS Microbiol. Rev. 31: 449-477; Taylor et al. 2011 Trends. Ecol. Evol. 26: 278-284 Gillings 2013 Frontiers in Microbiology 4: 4.
  • tetA,R Continued selection has assembled complex mosaic elements Plasmid NR1 Tn10 Tn9-like catA1 For example, plasmid NR1 contains DNA from as many as twelve different origins. Tn21 transposon backbone mer operon Tn402 intI1 IS1326 gene cassettessul Such molecules are xenogenetic, in the sense that they arise through human activity. IS1353 Gillings and Stokes 2012 Trends in Ecology and Evolution 27: 346-352.
  • There is good reason to suspect that the use of antibiotics is having effects beyond their intended role as therapeutic agents • • • • They affect non-target organisms in the human microbiome They are excreted unchanged, to affect environmental organisms They promote the fixation of complex, multi-resistance elements They have effects at sub-inhibitory concentrations
  • Too little diversity; high cost of maintenance Number of cells in population All mechanisms that generate diversity are under stabilizing selection; This balances the costs of maintaining genomic integrity against the potential benefits of genomic innovation Too much diversity; loss of genomic integrity Rate at which diversity is generated (by mutation, lateral transfer or recombination) Gillings and Stokes 2012 Trends in Ecology and Evolution 27: 346-352.
  • Number of cells in population Exposure to antibiotics, even at sub-inhibitory concentrations, induces the SOS response, causing transient increases in the rates of mutation, recombination and lateral gene transfer Transient increase in the overall rate at which diversity is generated Rate at which diversity is generated Mutation rates: Kohanski et al. 2010 Mol Cell 37: 311-320; Thi et al. 2011 Antimicrob Ag Chemo 66: 531-538 Recombination: Lopez & Blazquez 2009 Antimicrob Ag Chemo 53: 3411; Guerin et al. 2009 Science 324: 1034 Lateral transfer: Beaber et al. 2004 Nature 427: 72-74; Prudhomme et al. 2006 Science 313: 89-92
  • Number of cells in population Continual exposure to sub-inhibitory levels antibiotics is likely to select for cell lineages with inherently higher rates of mutation, recombination and lateral gene transfer Directional selection that favors lineages with inherently higher rates Rate at which diversity is generated Mutation rates: Desai and Fisher 2011 Genetics 188: 977-1014; Gentile et al. 2011 Biol. Lett. 7: 422-424 Recombination: Cambray et al. 2011 Mobile DNA 2: 6; Boucher et al. 2011 mBio 2: e00335-e410 Lateral transfer: Heuer et al. 2010 FEMS Microbiol Ecol 73: 190-196; Palmer & Gilmore 2010 mBio 1: e00227
  • Gillings and Stokes 2012 Trends Ecol. Evol. 27: 346-352
  • Test with two genome sequenced isolates: Ps. aeruginosa PA14 and Ps. fluorescens Pf5 Control Inoculate triplicate flasks Antibiotic 1 Antibiotic 2 Antibiotic 3 etc Serial passage Genome sequence 3 x single colony isolates from each experimental line: 3 isolates x 3 replicates x 6 treatments x 2 species = 108 genomes Compare with reference genome to score point mutations, transpositions, recombination events and indels.
  • To determine if sub-clinical levels of antibiotic pollution increase rates of interspecies lateral gene transfer, reference strains will be inoculated into soil mesocosms Control Inoculate triplicate mesocosms Antibiotic 1 Antibiotic 2 Antibiotic 3 etc Genome sequence 3 x single colony isolates from each experimental line: 3 isolates x 3 replicates x 6 treatments x 2 species = 108 genomes Compare with reference genome to score point mutations, transpositions, recombination events and indels.
  • We have access to a long term field trial (Ontario, Canada) where antibiotics have been applied each spring since 1999. Triplicate plots Control, low, medium, and high treatments Genome sequence 3 x single colony isolates of soil pseudomonads from each experimental line: 3 isolates x 3 replicates x 4 treatments x 2 species = 72 genomes Compare genomes to score point mutations, transpositions, recombination events and indels Topp et al 2013 J. Env. Qual. 42:173-178.
  • Transforming data to knowledge: • • • • • • • • Sequencing >290 x 6-7Mb genomes – platform? Storage of sequencing data Assembly and closure of high quality genomes High throughput pipeline for analysis of mutations Confirmation of mutational events Comparison of rates and statistical testing Calculation of effects on baseline rates Potential effects on molecular clock
  • Global Microbiome Pangenome Panproteome Mobilome Clinically important resistance genes Resistome Parvome Clinically important antibiotic molecules Clinically important resistance genes are a small sample of the resistome, just as clinically important antibiotics are a fraction of the small molecules made by bacteria. Effects wrought by antibiotics may influence the entire pangenome Gillings 2013 Frontiers Microbiol. 4: 4
  • Global Microbiome Pangenome Panproteome Mobilome Resistome Parvome Human antibiotic production Human synthesis of antibiotics overwhelms natural production, and large quantities are released into the environment. Because they are bioactive, pollution with antibiotics should be of serious concern, and classed in the same category as other xenobiotic compounds. Pruden et al. 2006 Eviron Sci Technol 40: 7445-7450; Storteboom et al. 2010 Environ Sci Technol 44: 1947-1953
  • Global Microbiome Pangenome Panproteome Mobilome Clinically important resistance genes Resistome Parvome Clinically important antibiotic molecules As selective pressures continue, more of the resistome will be recruited onto mobile elements, and the diversity of clinically important resistance genes will increase. General rates of mutation may increase across the entire microbiome. Gillings and Stokes 2012 Trends Ecol. Evol. 27: 346-352; Gillings 2013 Frontiers Microbiol. 4: 4
  • Paleoanthropocene Industrial Revolution Great Acceleration Resistance Pollution Evolvability Antibiotics Disinfectants Heavy metals Mercury Arsenic Human microbiome Shift to agricultural diet Dysbiosis Microbiomics Antibiotics Cesareans Bottle feeding Processed foods Black Death Dispersal/Disease Zoonoses Agricultural mutualisms Migration with parasites 11700 bp PLEISTOCENE 100,000 1775 HOLOCENE 10,000 1,000 years bp Vaccination Emerging disease Age of exploration Epidemics 1953 present ANTHROPOCENE 100 0 Antibiotic failure Pandemics