Water pollution by antibiotics andselection of antibiotic resistances

Fernando BaqueroDivision of Biology and Evolution of Microorganisms,

Department of MicrobiologyRamón y Cajal University Hospital

IRYCIS, CIBERESPMadrid, Spain

Water pollution by antibiotics andselection of antibiotic resistance

genes

Fernando BaqueroDivision of Biology and Evolution of Microorganisms,

Department of MicrobiologyRamón y Cajal University Hospital

IRYCIS, CIBERESPMadrid, Spain

THE GENOCENTRIC REVOLUTION

NOT the organisms,but

the GENES,as units for understanding

Nature

The organisms are just

VEHICLESfor the genes

Universe of genes from families of basic enzymes inall bacterial organisms, mostly environmental

In bacteria!

Acetyl-transferasesMethylasesNucleotidyl-transferasesEsterasesPhosphorylasesPeptidasesThiol-transferasesHydroxylasesGlycosyltransferasesOxydases….

Origin of antibiotic resistance genes

Universe of genesencoding basic enzymes

In bacteria!

Enzyme with somecoincidental antibioticdetoxification activity

Universe of genes encodingbasic enzymes

In bacteria!

Antibiotic exposure

Enzyme with somecoincidental antibioticdetoxification activity

Universe ofdifferent genesencoding basic

enzymes

In bacteria!

Selection of organismswith the natural enzyme

with coincidentalantibiotic detoxification

activity

Concentration high

Effective selection of antibiotic resistance at very small antibioticconcentrations

(Negri, Levin, Blázquez, Lipsitch, Baquero, AAC 2000); Baquero, Negri.Selective compartments for resistant organisms in antibioticgradients, Bioessays 1997)

Universe ofdifferent genesfrom a family ofbasic enzymes

In bacteria!

Climbing the hill

Under continuous exposure,climbing the hill to reach high

antibiotic resistance: fromsoft “coincidental functions”

to specialized antibiotic-resistance functions

Universe of genes encodingbasic enzymes

In bacteria!

Antibiotic exposure

Enzyme with somecoincidental antibioticdetoxification activity

• Might exposure be caused by microorganismsproducing antibiotics in nature?

• Might these organisms be selected by antibiotics?

Laskaris P, Tolba S, Calvo-Bado L, Wellington L. Coevolution ofantibiotic production and counter-resistance in soil bacteria.Environ Microbiol. 2010

The natural function of antibioticsin the microbial environment

Antibioticconcentration

Attack (kill to invade)?

Defense (kill the invaders)

Warning (Aposematism, discourage possible invaders)

Regulation (friendly coexistence, diversity)

Inhibitory concentration

Concentrationproducing any effect

Concentration Gradient

In nature antibiotic production is frequently a defensive strategy, not an aggressive one

Release of anthropogenic antibiotics disturbs thenormal bacterial semiotic space in the environment

Antibioticconcentration

Attack (kill to invade)?

Defense (kill the invaders)

Warning (Aposematism, discourage possible invaders)

Regulation (friendly coexistence, diversity)

Inhibitory concentration

Concentrationproducing any effect

Concentration Gradient

Semiotic space(antibiotics as signals)

PNAS 103:19484 (2006)

GeneGene

captureplatform

Mobilegeneticelement

Bacterialclone

Host(patient, carrier) Environment

Baquero et al., Public Health Evolutionary Biology of Antibiotic Resistance. Evolutionary Applications, 2015; Baez, delCampo, Baquero et al. Microb Drug Resist. 2015 (about Franklin Gulls)

Vehicles inside VehiclesA multilevel cascade of

transmissions andintrogressions

Biological entities are bothOccupants and Vehicles

Universe ofenvironmental

genes encodingbasic enzymes

In bacteria!

Under continuous exposure,climbing the hill to reach high

antibiotic resistance: fromsoft “coincidental functions”

to specialized antibiotic-resistance functions

Climbing the hill and transferto commensals and pathogens

Commensal

Pathogen

Resistance Gene Capture

Universe ofenvironmental

genes encodingbasic enzymes

In bacteria!

Under continuous exposure,climbing the hill to reach high

antibiotic resistance: fromsoft “coincidental functions”

to specialized antibiotic-resistance functions

Climbing the hill and transferto commensals and pathogens

Commensal

Pathogen

Resistance Gene Capture byMobile Genetic Elements

Plasmid Transfer

Capture of pre-resistance genes from wildorganisms and invasion of human

commensals-pathogens

CTX-M-2 gene captured from Kluyveraascorbata and mobilized by ISEcp1B

into Escherichia coli

A classic -10 years ago

Rowe-Magnus D, Mazel, D. The role of integrons in antibiotic resistance gene captureIJMM, 2002; Toleman, Bennett, Walsh: ISCR Elements: Novel Gene-Capturing Systemsof the 21st Century? MMBR 2006

K. ascorbata CTX-S

Gram -

Gram +

Genetransmissionvia plasmid

vehicles

Plasmid vehicles transmittingadaptive-accessory genes

(as Ab-R)

Lanza, V. F., Tedim, A., Martínez JL.,Baquero, F., & Coque, T. M. (2015).Microbiol Spectrum 3(1)

Svara, Rankin. BMCEvolutionary Biology 11:130

(2011)

cargo

Cargoinsidechassis

Gene(s)

Gene captureplatform

Plasmid, ICE

Cellular clone

Host microbiota

Environmental Microbial Communities

ChromosomePlasmids

Multilevel chain of captures andselections

JL Martínez, T Coque, F Baquero (2015)Nature Rev. Microbiol., 13:116-123

Ranking the Risks ofDetection of Resistance

Genes in Resistomes

Located in Mobile Genetic Elementsas Vehicles of Antibiotic ResistanceGenes play a Key-Role in the Risk

Classification

Involving R tonovel antibiotics

Involving R to antibioticsin clinical use

Involving R in high-riskhuman pathogens

JL Martínez, T Coque, F Baquero (2015) Nature Rev. Microbiol., 13:116-123

Transmission bottlenecks: quantities and horizontal gene transfer (HGT)

Resistantorganisms

JL Martínez, T Coque, F Baquero (2015) Nature Rev. Microbiol., 13:116-123

Transmission bottlenecks: quantities and horizontal gene transfer (HGT)

Resistantorganisms

Meeting reactors

Antibiotic therapy

HumanMicrobiota

AnimalMicrobiota

HospitalsLTCFs

FarmsAquaculture

Wastewater,effluents

Sewagetreatment

plants

Soil,Sediments

Surface,groundwater

Four Reactors for Antibiotic-Resistance Gene Transfer ((Baquero, Martínez and Canton, Current Op Biotechnol., 2008)

EnvironmentalBacteria

Antibiotic release

Where bacteria meet to exchange genes

Humic acids

Soil particles

Antibiotics

Bacterial cells

Particulated Sewage, Soil

Sewage, contaminatedwater, might contain bothbacterial and antibiotics

Modified from: Beredonk, TU et al., Tackling antibiotic resistance: the environmental framework. Nature Rev. Microbiol (2015)

AntibioticAggresion(Therapy)

Unwanted Aggression(Environment)

Back unwanted effects

Defense

Modified from;Kümmerer, K. (2009). Antibiotics in the aquatic

environment–a review–part I. Chemosphere, 75, 417-434.

ng/L

Pen: up to 0.2 mcg/ml

Mac: 0.7-4 mcg/ml

Quin: 0.1 mcg/ml

Sul: 1-2 mcg/ml

Tet: up to 0,02 mcg/ml

Sewage treatment plant effluent Surface water

Tmp: up to 0.5 mcg/ml

Chl: 0.1-0.5 mcg/ml

0.01-0.5

0.03-0.1

0.02-0.4

0.02-0.04 Antibiotic concentrations in water

Gullberg E, et al. (2011) Selection of Resistant Bacteria at Very Low Antibiotic Concentrations. PLoS Pathog 7(7)

1:100 MIC

1:4 MIC

Selection at Subinhibitory Antibiotic Concentrations

Growth ratesare reduced well

below MICSelection

Baquero F, Negri MC. Selective compartments forresistant microorganisms in antibiotic gradients.Bioessays. 19:731-6 (1997)

Environmental Pharmacology of Antibiotics(PK/PD)

Jechalke S, Heuer H,Siemens J, AmelungW, Smalla K. Fate andeffects of veterinaryantibiotics in soil.Trends in Microbiol22:536, 2014

Pal C. eta al. The structure anddiversity of human, animal andenvironmental resistomes.Microbiome 4:54, 2016

Wastewater-sludge

Surface water

Gastrointestinal

Antibiotic-R genes Biocide-Metal-R genes Mobile Genetic Elements

R-gene capture platformsin microbiome

Based on SeqCap EZ

Fernandez-Lanza,Coque, Baquero,Bioinformatics 2016

Reads/kb (normalized per genesize)

ResCap and Conventional Metagenomics in the detection of Antibiotic Resistance Genes

ResCap versus conventional metagenomics: abundance anddiversity of biocide-resistance putative genes

ResCap versus conventional metagenomics: abundance anddiversity of relaxase genes (plasmid type markers)

Release of anthropogenic antibiotics in soil:disturbing a complex signaling network

• Antibiotics which can be used by bacteria in sub-inhibitoryconcentrations for signaling and maintenance of complexregulatory networks can have short-term and long-termimpacts on the structure and function of soil bacterialcommunities if applied with manure in high concentrations.

Jechalke S, Heuer H, Siemens J,Amelung W, Smalla K. (2014).Fateand effects of veterinary antibioticsin soil. Trends in Microbiol 22:536

BrunoGonzalez-Zorn

Fernandode la Cruz(CantabriaUniversity)

(UCM)

Jose LuisMartínez(CNB, CSIC)

Ricardo Ramos (PCM)

Fish Sewage

Living bacteria and all what they contain

What are “antibiotic resistance genes” in a publichealth perspective?

• 3-5 % of all genes in a given bacteria might directly or with simplemutational changes increase the MIC to one or more antimicrobialagents, frequently producing low-level (but eventually selectable)resistance.

• Most of these genes belong to the house-keeping functions of bacteria (as pumps, ortopoisomerases, or PBPs), providing resistance in the exposed bacterial hosts, butnot beyond.

• Resilience genes: mainly chromosomal genes which assures(eventually mutated) the maintenance of the host bacteria in theirenvironment, when antibiotics are present. Not “born” resistance genes!

• Resistance genes: genes with a predominant function on antibioticdetoxification, with potential clinical consequences, frequentlysubmitted to spread in bacterial populations by horizontal genetransfer.

Baquero F, Negri MC. Selective compartments for resistant microorganisms in antibiotic gradients.Bioessays. 19:731-6 (1997)

Complexity of antibiotic gradients

Changing in time Merging gradients

Heterogeneity ofsources

Martínez & Baquero, Mutation Frequencies andAntibiotic Resistance. AAC 44:1771 (2000)

Subinhibitory concentrations selectfor many low-level, low-specificresistant mutants

Inhibitory concentrations select forfew high-level, highly specificresistant mutants

Low level resistance

might assure survivalto evolve

High level resistance

Mutation

Horizontal genetransfer

Competition experimentsbetween susceptible andresistant strains underciprofloxacin exposure

Gullberg E, et al. (2011) Selection of Resistant Bacteria at VeryLow Antibiotic Concentrations. PLoS Pathog 7(7): e1002158.

Ciprofloxacin concentrationsGenerations of Growth

Type of resistance mutants

MSC: Minimal Selective Concentration

1:10 MIC

1:230 MIC

1:10 MIC

1:10 MIC

Sublethal Antibiotic Treatment Leads to MultidrugResistance via Radical-Induced Mutagenesis

Kohanski, De Prisco, Collins(2010) Molecular Cell 37:311-320,

Gullberg E, et al. (2011) Selection of Resistant Bacteria at Very Low Antibiotic Concentrations. PLoS Pathog 7(7)

1:100 MIC

1:4 MIC

Competition Experiments: Selection at Subinhibitory Antibiotic Concentrations

Selection of a strain harboring a costly resistance plasmidby sub-inhibitory concentrations of antibiotics

The plasmid imposes 4% of fitness cost(reduction in bacterial replication)

Gullberg E et al., (2014) mBio 5e1918-14

Selection of a strain harboring a resistance plasmidby sub-inhibitory concentrations of antibiotics

The plasmid imposes 4% of fitnesscost (reduction in bacterial replication)

Gullberg E et al., (2014) mBio 5e1918-14

Selection of a strain harboring a resistance plasmidby sub-inhibitory concentrations of metals

The plasmid imposes 4% of fitnesscost (reduction in bacterial replication)

Gullberg E et al., (2014) mBio 5e1918-14

Selection of a strain harboring a resistance plasmidby sub-inhibitory concentrations of antibiotics

The plasmid imposes 4% of fitnesscost (reduction in bacterial replication)

Gullberg E et al., (2014) mBio 5e1918-14

Plasmids harboring antibiotic resistanceseven with a high fitness cost can be

maintained in a bacterial population byan infinitesimally low concentration of

the antibiotics, or metals

Examples of measured fluoroquinolone concentrations

Sewage water:Sweden 0.1-0.3 ng/ml (Water Research 41:613)Spain 1-2 ng/ml (Water Research 69:234)China 0.7-5 ng/ml (Chemosphere 119:1379)

1- to 50-fold above minimal selective concentration

Hospital effluent:Sweden 2-14 ng per ml (Report County Council Uppsala 2005)Spain 14-20 ng per ml (Water Research 69:234)

20- to 200-fold above minimal selective concentration

Assuming that the initial fraction of a resistant strain (gyrA) is 1/100 how long does it take for it to outcompete the susceptible strainand become the dominant population at the above fluoroquinolone levels?

Generation time of bacteria1 hour 10 hours 100 hours

2 ng/ml 3.3 days 33 days 330 days

5 ng/ml 0.8 day 8 days 80 days

Time required for resistant mutants to become enriched

Dan Andersson, Personal Communication after publishing Gullberg et al PLOS Pathogens 2011 and mBio 2014

Baquero F, Coque TM. (2014)MBio. 2014 Dec 9;5(6):e02270

A local increase in antibioticconcentration produces a field of

subinhibitory concentrations; the “spaceof selection” is proportional to theintensity of the antibiotic release

On the right,under selection

Minimal Bactericidal Concentration

Minimal Inhibitory Concentration

Minimal Plasmid Maintenance Concentration

Minimal Antibiotic Concentration

Spaces of Antibiotic Selection

“Fighting, defeating antibiotic resistance”A naïve statement.

Leslie Orgel’s Second Rule:

"Evolution is cleverer than you are.“

• We have 1012 E. coli cells in the intestine of a single humanhost, with about 200,000 mutations per gene and day.

• A gram of soil may contain between 5,000 and 40,000 speciesof microbes

• “Modular engineering of resistance”:

Transposases are the most common and abundant genesin nature.

Unlimited offer of bacterial genetic variation andengineering in an unlimited number of niches

Impossible to prevent theemergence of antibiotic

resistance genes

Sick waterbecause ofantibioticresistance

Antibiotic resistance notmentioned in the document!

United Nations Environment Programme (UNEP)- UN HABITAT

antibiotics were initially derived (Dantas et al., 2008; D’Costa et al., 2006;

Riesenfeld et al., 2004). In naïve bacterial populations, “resistance” genes are

likely to encode other functions (e.g., metabolism, regulation) that nevertheless

offer a selective advantage, Davies explained. “Resistance genes in the environment,

in general, are not resistant,” he said. “They become resistant when picked

up and overexpressed in a foreign cytoplasm.”

Opportunities for such acquisitions are presented by the flow of water

among the various environments in which bacterial resistance genes exist, Davies

observed. In particular, wastewater treatment plants—which he described as “an

incredible mixing pot of genes and plasmids”—provide an ideal opportunity for

pathogenic bacteria to acquire new resistance genes, and new virulence genes as

well (see Davies in Chapter 4). He noted recent studies by Szczepanowski and

coworkers, who isolated and sequenced antibiotic-multiresistant plasmids from

bacteria present in sludge in wastewater treatment plants, and found that they also

contained several virulence-associated genes and integrons (Szczepanowski et al.,

2004, 2005). Such plasmids, moreover, were detectable in effluents released from

the treatment plant into the environment (Szczepanowski et al., 2004). Researchers

from the same laboratory have also performed a metagenomic analysis of such

bacteria and determined that their collective plasmid DNA encoded resistances to

all major classes of antimicrobial drugs (Szczepanowski et al., 2008).

The pervasiveness of antibiotic resistance in the environment suggests that

antibiotics—that is, molecules with antibiotic activity—are equally abundant in

nature, produced by bacteria (and also by plants) to serve a variety of purposes,

Davies said. Thus, to find novel antibiotics, his laboratory is pursuing a strategy

of identifying organisms that produce bioactive compounds, then analyzing

these compounds for their antibiotic properties. Similarly, Handelsman (see

Chapter 4) described a process by which she and coworkers are searching the soil

Environmentalsources ofantibioticresistance genesand antibiotics