introduction - clostridia課件

LisaDrummondUniversityofEdinburgh AntibioticsandClostridiumdifficile Introduction Grampositivespore formerobligatelyanaerobicfirstdescribedinasymptomaticneonatesincreaseduseofantibioticsledtoanincreaseinC difficiledisease Introductioncont infectionrangesfromasymptomatic milddiarrhoea colitistopseudomembranouscolitisriskfactors antibiotics age environmentandvirulenceofinfectingstrainthirdgenerationcephalosporins clindamycinandamoxycillinassociatedwiththegreatestriskdiseaseoccursafterdepletionofpatient snormalprotectiveflora Diseaseprocess ANTIBIOTICTHERAPY ALTERATIONOFCOLONICMICROFLORA C difficileEXPOSURE COLONISATION RELEASEOFTOXINA TOXINB COLONICMUCOSALINJURYANDINFLAMMATION AdaptedfromKellyCP LaMontJT 1998 Clostridiumdifficileinfection AnnualReviewofMedicine49 375 390 IncidenceofC difficileinthepopulation AdaptedfromKellyCP LaMontJT 1998 Clostridiumdifficileinfection AnnualReviewofMedicine49 375 390 PathogenicityLocus PaLoc 19 6kbelementreplacedby115bpinnon toxigenicstrainstcdDalternativesigmafactortcdCputativenegativeregulatortoxinstranscribedonentrytostationaryphase PaLoccont toxinproductionaffectedbyglucose sub inhibitoryconcs ofantibiotics aminoacids temperature oxidativestress biotininsufficiency biocarbonateconcentration AIMS toanalyseMICdata patientantibioticregimes S types resistancetolookateffectsofsub MICsongrowthandtoxinproductioninvestigatetoxintranscriptsusingRT PCRinvestigatetotalcellproteinbetweencontrolsandsub MICantibioticsusing2DgelelectrophoresisandMALDI TOF MICs 186strainsand6antibiotics NCCLS thetwotreatmentagents vancomycinandmetronidazole4precipitatingagents amoxycillin clindamycin cefoxitinandceftriaxonedatabaseutilisedforanyconnections Clindamycinresistance 12isolatestestedhadclindamycinMICof 128 g mlallcontainedermBgene2differentsizesnotedsmallerbandlackleaderpeptide Farrowetal 2002 Recurrencesandreinfections somepatientsproducedupto12samplesoverthe18monthsallowedcomparisonsoftheirisolatesoverthattimesomepatientshadchangingS typesoverthistimesomepatientsalsohaddifferentisolatesinthesamefaecalsample MICconclusions nostrainsresistanttovancomycinormetronidazolenosignificantdifferenceofresistanceprofilesbetweenS typesnocorrelationbetweenantibioticsgivenandresistanceprofilesevidenceofmixedinfectionsorrecurrences Sub MICantibiotics antibioticshavepreviouslybeenshowntoaffecttoxinproductioninC difficilevastamountsofliteratureshowingeffectsonotherbacteriathoughthere sverylittledataforC difficile Sub MICexperimentalset up usedsame6antibioticsasMICworkusedreferencestrainNCTC11223 locallyendemicstrain338aandsequencedstrain6301 2 1 4and1 8sub MICconcs usedsampled3Xadayfor104hoursOD600measuredeachtimeand1mlofsupernatefrozenforELISAanalysis Controlsfromsub MICexperiments eachstraingrown6timesintotalgrowthvariedlittlebetweenstrainstoxinelaboratedatslightlydifferenttimesinthegrowthcurvetoxinproductionby338aand630exceedsassaybyca 48h Sub MICconclusions there softenalaginthegrowthofthebacteriacomparedtothecontrolmaineffectontoxinisthatit selaboratedquickerundersub MICconditionsheterogeneitycommonbetweenstrainsfortoxinproductionandgrowthinresponsetoantibiotics RT PCR wantedtolookfortoxintranscriptstoseeiftheycorrelatetosub MICworkRNAconcentrationslow ca 5 g ml 16StranscriptseasilyseenbutonlywithSensiscriptenzymelowconcentrationsofRNAprobablymadetoxintranscriptsdifficulttosee Sensiscript Sensiscriptvastlyimprovesabilitytopickup16SRNAstillnotranscriptsfromtoxinsdecidetocutlossesastimeextremelyshort RT PCRoutcome Wasunsuccessfulinseeingtranscriptsfortoxins tcdC tcdDandgroELuseofSensiscriptledtoclearsignalfrom16SRNAifhadmoretimewouldhavetriedanothertechniquee g Trizol Trireagentetc Proteomics use2DgelelectrophoresisandMALDI TOFanalysisofproteinsproteinprofilestilllargelyuncharacterisedinC difficilewantedtocomparecontrolvs sub MICsamplepreparationreproducibilitynewMASCOTdatabasebeingset up Controlvssub MIC gelsveryreproducible goodforfuturemanipulationsnoobviousdifferencebetweentwosetsofconditions withandwithoutceftriaxone 40spotsfrom6gelsweretakenforMALDI TOFdatastillbeinganalysedandnewMASCOTdatabaseinthepipeline Typical2Dgel Conclusions MICs nostrainsresistanttoeitherofthetreatmentagentsnosignificantdifferenceofresistanceprofilesbetweenS typesnocorrelationbetweenantibioticsgivenandresistanceprofilesevidenceofmixedinfectionsorrecurrences Conclusions sub MICs sub MICantibioticsoftencauseagrowthlagandshiftforwardtheproductionoftoxinthereisheterogeneitybetweenstrainsandtheirresponsetosub MICantibioticstheeffectontoxincouldnotbeseenmirroredinthetoxintranscriptsduetothesensitivityoftheRT PCR Conclusions proteomics reproducibility goodsamplepreparationthecombinationofstrain630andceftriaxoneproducedaproteinprofileunchangedtothatofthecontrolonc