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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