The Impact of Antibiotics on the Gut Microbiota.docx

The Impact of Antibiotics on the Gut Microbiota.docx

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TheImpactofAntibioticsontheGutMicrobiota

TheImpactofAntibioticsontheGutMicrobiotaasRevealedbyHighThroughputDNASequencing

PublishedonMarch15,2012

Author:

 PaulD.Cotter

Specialty:

 Microbiology, Antimicrobials, GutMicrobiota

Institution:

TeagascFoodResearchCentre

Address:

Cork,Ireland 

Institution:

AlimentaryPharmabioticCentre 

Address:

Cork,Ireland

Author:

 CatherineStanton

Specialty:

 Microbiology, Probiotics, Prebiotics, InfantHealth

Institution:

TeagascFoodResearchCentre

Address:

Cork,Ireland 

Institution:

AlimentaryPharmabioticCentre 

Address:

Cork,Ireland

Author:

 R.PaulRoss

Specialty:

 Microbiology, Antimicrobials, Probiotics

Institution:

TeagascFoodResearchCentre

Address:

Cork,Ireland 

Institution:

AlimentaryPharmabioticCentre 

Address:

Cork,Ireland

Author:

 ColinHill

Specialty:

 Microbiology, Antimicrobials, FoodPathogens

Institution:

DepartmentofMicrobiology,UniversityCollegeCork

Address:

Cork,Ireland 

Institution:

AlimentaryPharmabioticCentre 

Address:

Cork,Ireland

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Abstract:

 Treatmentwithbroadspectrumantibioticscanhaveadetrimentalimpactonthecommensalbacteriapresentinthegut.TheextensivenatureofthecollateraldamagecausedbysuchcompoundshasbeenrevealedmorestarklythaneverbeforethroughtheapplicationofhighthroughputDNAsequencing-basedtechnologiestoinvestigateresultingmicrobialpopulations.Herewereviewthefindingsofsuchstudiesanddiscussthestrategiesavailabletominimizesuchnegativeimpacts.

Introduction

Thediscoveryof antibiotics intheearlytwentiethcenturyisarguablythesinglemostsignificantbreakthroughtopositivelyimpactonhumanhealthinrecenthistory.Indeed,human lifeexpectancyjumpedbyeightyearsintheperiodbetween1944and1972,afactwhichismainlyaccreditedtotheintroductionofantibiotics.Thedevelopmentofmicrobialresistanceisthenegativeconsequencemostfrequentlyassociatedwiththeoveruseofantibiotics.However,thecollateraldamagewhichtheseantimicrobialsinflictonbeneficialgutmicrobialpopulations（andothercomponentsofthehumanmicrobiota）couldbeamuchmoresignificantproblem（Blaser,2011）.Thelongtermhealtheffectsofantibiotictreatmentinearlylifearenotwelldefinedbut,notably,antibiotic-mediatedchangestothecommensalmicrobiotahavebeenassociatedwiththedevelopmentof asthma,eczema, atopicdermatitis andotherallergicsensitization, autoimmuneencephalitis,candidiasis,cholera,andpathogeninducedcolitis（Willing etal.,2011）.Collateraldamagetocommensalmicrobesresultsfromthefactthatthemajorityofantibioticsemployedroutinelyhaveabroadtargetrange.Thereareobviousbenefitsassociatedwiththeuseofbroadspectrumantimicrobialswhentreatingsevereinfectionsofunknownorunclearetiology.However,evenincaseswhentherelevantdisease-causing（pathogenic）bacteriaisidentified,broadspectrumantibioticsarealsomostcommonlyemployedasaconsequenceoftheabsenceofnarrowspectrumalternatives.Thisisatleastpartiallyduetothefactthatthe pharmaceuticalindustry focusesprimarilyonthedevelopmentofbroadspectrumantibioticswhichcanbeemployedtotreatavarietyofdifferentinfections.Whilebroadspectrumantibioticsmayinhibitseveralpathogenicbacteria,thiskillingactioncanalsoextendtoincludecommensalgutbacteriathatcontributetohumanhealth.However,itisonlyinrecentyearsthroughtheadventofhighthroughputDNAsequencingtechnologiesthatthefullextentoftheimpactofantibioticuseonthe gutmicrobiota hasbecomeapparent.Throughadeeperunderstandingofthenegativeconsequencesofantibioticadministration,itishopedthatthemechanismsbywhichthesechangescontributetoillhealthcanbebetterunderstoodandstrategiescanbedevelopedtoensurethatthecollateraldamageinflictedisminimal.

TheImportanceoftheGutMicrobiota

Althoughfrequentlyquotedin“humanmicrobiota”-relatedpublications,thefactsandfiguresrelatingtothenumbersandfunctionsof commensalbacteria,andthoseinthegutinparticular,remainawe-inspiring.Thehumanmicrobiotaconsistsofapproximately1014 bacterialcells,10-foldhigherthanthetotalnumberofcellsinahumanbody（Ley etal.,2006）.Ofthemicrobialpopulationsassociatedwiththehumanbody,thelargestandmostcomplexisthatpresentinthegut（orgastrointestinaltract）.Indeed,withinthegut,thelargeintestinealoneharbors1010-1011 bacterialcells/g.Arecentinvestigationoffecalsamplesof124Europeansrevealedamicrobiota-associatedgenesetwhichisapproximately150timeslargerthanthehumanhostgenerepertoire.Itwasalsonotablethatexaminationofthecombinedcohortrevealedbetween1,000-1,150prevalentbacterialspecies（Qinetal.,2010）.Theinvestigationofthesegut-associatedbacteriaiscomplicatedbythefactthatthemajorityareyettobegrowninthelaboratory（Goodman etal.,2011）andithasonlybeenthroughthedevelopmentofcultureindependentapproaches,andmostrecentlythroughtheapplicationofhighthroughputsequencing,thatithasbecomepossibletogainanunbiasedinsightintothecompositionofthesepopulations.

TheApplicationofHighThroughputDNASequencing-basedTechnologiestoStudytheGutMicrobiota

WhileanumberofhighthroughputDNAsequencingplatformsexist（Glenn,2011）,theyaretypicallyappliedtostudyingbacterialecosystemsineitheroftwoways:

todeterminethebacterialcompositionoftheenvironmentthroughsequencingofthe16srRNAgene,ortodeterminethefunctionalpotentialofthebacterialpopulationthroughshotgun（random）sequencingofDNA（Petrosino etal.,2009）.Theformerstrategyhasbeenusedtoassesstheimpactofantibioticsongutbacteria.Itreliesonthefactthatthe16srRNAgeneispresentinallbacteriaandcontainsconservedandvariableregionswhichfacilitatethegenerationofampliconsusingdegenerate PCR primers.ThisallowsforthesubsequentidentificationofthebacteriapresentonthebasisofDNAsequencedifferencesintheseamplicons.ThesameprincipleunderliesthesequencingofclonelibrariesexceptthatintheseinstancesthePCRampliconsarefirstclonedintoavectortofacilitatesequencingusingtraditional,lowthroughput（Sanger）sequencing.Whiletherehavebeenanumberofimportantpublicationsinrecentyearsinwhichtheimpactofantibioticsonthegutmicrobiotahasbeenassessedusingclonelibraries[suchasthoseinvestigatingtheimpactofdifferentβ-lactamsormacrolidesonthegutmicrobiotaofadulthumans（Morotomi etal.,2011）orthoseassessingtheinfluenceofstreptomycin（Garner etal.,2009）, vancomycin （RobinsonandYoung,2010）oracombinationofbacitracin,enrofloxacin,and neomycin sulphateontheentericmicrobiotaofmice（Puhl etal.,2011）],herethefocuswillspecificallybeonthosewhichhavebenefitedfromthemuchgreaterdepthofsequencinginformationprovidedbyhighthroughputapproaches.

HighThroughputDNASequencing-basedInsightsintotheImpactofAntibioticsontheGutMicrobiota

Despitethedramaticadvancesthathavebeenmadeinrecentyearsduetotheapplicationofhighthroughputsequencingtechnologiestothecharacterizationofgutmicrobialpopulations,therearerelativelyfewinstancesinwhichthistechnologyhasbeenappliedtoassessingtheimpactofantibioticadministrationontheentericmicrobiota（Table1）.Thosewhichhavebeenperformedhavefocusedonhuman（including exvivo）,canine,andmurinemicrobialpopulations.Theyhavealsovariedinthat,insomeinstances,thefocusofattentionisontheimpactofasingleantibiotic,whileinotherinstancesacocktailofantibioticsisemployedand,inoneinstance,theimpactofantibioticadministrationwasassessedwithoutregardfortheactualtherapeuticagent（Table1）.

Inthislattercase（Claesson etal.,2011）,thestudyinvolvedaninvestigationofthegeneraloverallimpactofantibioticadministrationonthegutmicrobialcompositionof43individualsinreceiptofantibiotics,relativeto118individualswhowerenot.ItrevealedthatthoseindividualswhohadreceivedantibioticshadgutmicrobialpopulationsthatconsistedofrelativelygreaterproportionsofBacteroidetes （andtheassociated Parabacteroides species）andreducedproportionsof Firmicutesand Proteobacteria.Thegutmicrobiotaof26oftheindividualswhohadnotbeeninreceiptofantibioticsattime0（T0）wassubsequentlyreassessedfollowingathreemonthinterval.Duringthisperiod,5ofthese26hadbeenadministeredantibioticsandcomparisonofthedatafromT0 andT3months revealedthatantibioticadministrationwasassociatedwithasignificantreductionintherelativeabundanceofbacteriafromthephylum Actinobacteria （Claesson etal.,2011）.

Assessmentoftheimpactofantibioticcocktailsrelativetothatofspecificantibiotics

Theotherstudiesassessingthenetimpactofantibioticadministration,asassessedthroughtheadministrationofacocktailofantibioticstomice,havealsoprovidedvaluableinsights.Inoneinstance,theimpactofacocktailof metronidazole,vancomycin,andneomycin,relativetothatofvancomycin（aglycopeptide）or ampicillin （β-lactam）alone,onthegutmicrobiotaofmicewasexamined（Ubeda etal.,2010）.Itwasnotedthatallthreetreatmentsreducedthenumberof16SrDNAcopies（representativeoftotalbacterianumbers）byafactorof100intheileumwhereasonlyampicillinconsistentlyreducedthenumberinthececum.Allantibiotictreatmentsdramaticallyalteredtheilealandcecalmicrobiota.Ampicillintreatmentledtoincr