Xenobiotics and Xenogenetics: Evolutionary consequences of

antibiotic use

Michael Gillings

Department of Biological Sciences and Genes to Geoscience Research Centre, Macquarie University

michael.gillings@mq.edu.au

Humans are the world’s greatest evolutionary force

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

Palumbi 2001 Science 293: 1786-1790

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?

Antibiotic ResistanceIs arguably the outstanding example of evolution by natural selection

Mutation and lateral transfer of genes between cells and species drives this phenomenon

Selection in the human Microbiome

Antibiotics select for mutations and lateral transfer events that confer resistance. Large quantities of antibiotic are excreted unchanged.

Humans create environmental hotspots for bacterial evolution

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.

Selection drives fixation of complex DNA elements

Tn402

intI1

gene cassettes

mer operon

transposon backbone

Tn21

IS1326

IS1353

Tn9-like

Plasmid NR1

Tn10

sul

tetA,R

catA1

Gillings and Stokes 2012 Trends in Ecology and Evolution 27: 346-352.

Continued selection has assembled complex mosaic elements

For example, plasmid NR1 contains DNA from as many as twelve different origins.

Such molecules are xenogenetic, in the sense that they arise through human activity.

Unintended effects of antibiotic useThere 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

Effects on basal rates of evolutionAll mechanisms that generate diversity are under stabilizing selection; This balances the costs of maintaining genomic integrity against the

potential benefits of genomic innovation

Rate at which diversity is generated (by mutation, lateral transfer or recombination)

Too much diversity; loss of genomic integrity

Too little diversity; high cost of

maintenance

Num

ber

of c

ells

in p

opul

atio

n

Gillings and Stokes 2012 Trends in Ecology and Evolution 27: 346-352.

Antibiotics induce the SOS responseExposure to antibiotics, even at sub-inhibitory concentrations, induces the

SOS response, causing transient increases in the rates of mutation, recombination and lateral gene transfer

Rate at which diversity is generated

Num

ber

of c

ells

in p

opul

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n

Transient increase in the overall 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

And select for evolvabilityContinual exposure to sub-inhibitory levels antibiotics is likely to select for cell lineages with inherently higher rates of mutation, recombination and

lateral gene transfer

Rate at which diversity is generated

Num

ber

of c

ells

in p

opul

atio

n

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

Directional selection that favors lineages with inherently higher rates

Key Research Questions

Gillings and Stokes 2012 Trends Ecol. Evol. 27: 346-352

in vitro experimentsTest with two genome sequenced isolates:

Ps. aeruginosa PA14 and Ps. fluorescens Pf5

Inoculate triplicate flasks

Control 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.

Soil mesocosm experiments

Inoculate triplicate

mesocosms

Control 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.

To determine if sub-clinical levels of antibiotic pollution increase rates of interspecies lateral gene transfer, reference strains will be

inoculated into soil mesocosms

Field experiments

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 genomesCompare genomes 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

Topp et al 2013 J. Env. Qual. 42:173-178.

Linking genomic data with eco-evo questions

• 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

Transforming data to knowledge:

Global Microbiome

Pangenome Panproteome

Parvome

Resistome

Mobilome

A conceptual map of the microbial world

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

Clinically important resistance genes

Clinically important antibiotic molecules

Global Microbiome

Pangenome Panproteome

ParvomeResistome

Mobilome

Human antibiotic

production

Antibiotics as pollutants:

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

Parvome

Resistome

Mobilome

Clinically important resistance genes

Clinically important antibiotic molecules

The Future:

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

100,000 10,000 1,000 100 0years bp

HOLOCENEPLEISTOCENE ANTHROPOCENE

11700 bp 1775 1953

Human microbiomeShift to agricultural diet

Processed foods AntibioticsCesareansBottle feeding

DysbiosisMicrobiomics

Dispersal/Disease

Resistance MercuryArsenic

AntibioticsDisinfectantsHeavy metals

PollutionEvolvability

present

Migration with parasites

Zoonoses Agricultural mutualisms

Age of explorationEpidemics

Black Death

VaccinationEmerging disease

Antibiotic failurePandemics

Paleoanthropocene Industrial Revolution Great Acceleration

Human effects on the global microbiome: commensals and

pathogens