Theproliferationofantibioticresistantpathogenshasincreasedattentiontotheuseofdrugcombinationstocombatthe evolutionofresistance.However,whilethephenotypicandgenotypicevolutionarypathstowardsmultidrugresistancecan be constrained by tradeoffs between resistance to one drug and susceptibility to other drugs or the humanhost environment, evolution often circumventstheseobstacles.FocusingonbothE.coliandclinicalspecies,wewillunravel the potential and limits of such approaches for constraining evolution.
In Aim 1, using a novel selection device, the MEGAplate,whichfollowsmultiplediversifyingbacteriallineagesastheymigrateandevolveonlargeantibioticgradient landscapes, we will comprehensively map the repertoireofmultimutationalpathstohighlevelresistancetoarangeof antibiotics as well as to pairsofantibioticspresentingadaptivetradeoffs.Coupledwithwholegenomesequencingand automatedhighthroughputphenotyping,thisdevicewillenableustoidentifycommonandspecificadaptivemechanisms, revealthepredictabilityandfurtherevolutionarypotentialofeachmutationalstep,andtestwhetherchannelingevolution towards ?quasi deadend? genotypes can impede longterm adaptation.
In Aim 2, we focus on cycling drug pairs presentingreciprocaladaptivetradeoffsandexaminevulnerabilitiesoftheapproach.Going?beyondtheaverage?,wewill construct a deep library of singlestep mutants selected on one of several different antibioticsandtestenmassetheir crossresistance to the other antibiotics. These results will identify rare ?escape? mutants which circumvent inherent tradeoffs between resistance to these drugs. Synthetically combining pairs of mutations which individually show resistance to one drug yet sensitivity to the other, we will reveal whether such mutations can interact nonadditively leadingtoresistancetobothdrugs.
In Aim3, weexaminetheeffectivenessyetweaknessesofnew?selectioninverting? compounds which we recently found to act preferentially against bacteria expressing the tetracycline resistance efflux pump. We will use a microfluidic device to follow single cells while switching between tetracycline and the new compounds,therebyidentifyingtheoptimalregimeforselectionagainstthepump.Wewillthensystematicallymutatethe tetracyclinepumptoidentifymutationsthatescapethetradeoff.Finally,inAim4,wefocusonthedifferencesbetweenin vitroandinvivoadaptivepathwaystoidentifytheevolutionaryconstraintsimposedbythehumanhostenvironment.We will use longitudinal isolates from clinical outbreakstoidentifywhichoftheresistantmutationsobservedinthelabalso appearduringpathogenevolutionwithinthehumanbodyandwhichothersareinvivoinaccessible.Comparingevolution of pathogens in immunocompetent versusimmunosuppressedpatientswillpointtoevolutionaryconstraintsimposedby innate immunity. In summary, our proposed research will reveal the genotypic and phenotypic constraints that govern evolutionary pathways towards multidrug resistance, both in the lab and during longterm infection in humans. Our longtermgoalistohelpdesigndrugregimesthatbetterpreventtheemergenceofresistanceandtodevelopalgorithms which based on the database of observedmutationalpathswillallowgenomebaseddiagnosticsofmicrobialinfections thatcanbothpredictcurrentresistanceprofileandanticipateitsfutureevolution.

Public Health Relevance

Relevance to public health: Antibiotics are the most direct and effective approachavailable against many infectious diseases, but their usefulness is being undermined by the spread of drugresistantpathogens.Weproposetostudydrugcombinationsthatmayconstrainandslow down the spread of drugresistancewhilestillprovidingeffectivetreatmenttocombatdisease. Beyond laboratory experiments, this study is also designed to determine how bacterial pathogens become resistant to many drugs during the course of a clinical infection, with the goalofprovidingtoolstoslowtheevolutionofresistance.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM081617-11
Application #
9478220
Study Section
Genetic Variation and Evolution Study Section (GVE)
Program Officer
Janes, Daniel E
Project Start
2007-07-02
Project End
2019-05-31
Budget Start
2018-06-01
Budget End
2019-05-31
Support Year
11
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Technion-Israel Institute of Technology
Department
Type
DUNS #
600133854
City
Haifa
State
Country
Israel
Zip Code
32000
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Russ, D; Kishony, R (2018) Additivity of inhibitory effects in multidrug combinations. Nat Microbiol 3:1339-1345
Chung, Hattie; Lieberman, Tami D; Vargas, Sara O et al. (2017) Global and local selection acting on the pathogen Stenotrophomonas maltophilia in the human lung. Nat Commun 8:14078
Schultz, Daniel; Palmer, Adam C; Kishony, Roy (2017) Regulatory Dynamics Determine Cell Fate following Abrupt Antibiotic Exposure. Cell Syst 5:509-517.e3
Lieberman, Tami D; Wilson, Douglas; Misra, Reshma et al. (2016) Genomic diversity in autopsy samples reveals within-host dissemination of HIV-associated Mycobacterium tuberculosis. Nat Med 22:1470-1474
Kelsic, Eric D; Chung, Hattie; Cohen, Niv et al. (2016) RNA Structural Determinants of Optimal Codons Revealed by MAGE-Seq. Cell Syst 3:563-571.e6
Stone, Laura K; Baym, Michael; Lieberman, Tami D et al. (2016) Compounds that select against the tetracycline-resistance efflux pump. Nat Chem Biol 12:902-904
Chait, Remy; Palmer, Adam C; Yelin, Idan et al. (2016) Pervasive selection for and against antibiotic resistance in inhomogeneous multistress environments. Nat Commun 7:10333
Baym, Michael; Lieberman, Tami D; Kelsic, Eric D et al. (2016) Spatiotemporal microbial evolution on antibiotic landscapes. Science 353:1147-51
Bairey, Eyal; Kelsic, Eric D; Kishony, Roy (2016) High-order species interactions shape ecosystem diversity. Nat Commun 7:12285

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