poster abstract book...1 poster numbers poster number presenting author abstract title page number...

@MicrobioSoc#Anaerobe19

microbiologysociety.org/Anaerobe19

FOCUSED MEETING

2019

Anaerobe 2019: Changing perceptions of anaerobic bacteria; from pathogen to the normal microbiota and back

13–14 June 2019 Jurys Inn, Cardiff, UK

POSTER ABSTRACT BOOK

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PosterNumbersPosterNumber PresentingAuthor AbstractTitle

PageNumber

01

DavidNygren AnationwideretrospectivestudyofinvasiveinfectionswithFusobacteriumnecrophoruminSweden2010-2017

3

02DafyddThomas Improvingthesurveillanceofantimicrobial

resistancetrendsamongstanaerobicoralpathogens

4

03KathleenBoiten Prevalenceofresistancegenesamongless

knowngram-negativeanaerobicbacteria,isolatedfromclinicalhumaninfections

5

04KathleenBoiten Discoveringanovelresistancegenefor

carbapenemresistanceinParabacteroidestimonensisusingWholeGenomeSequencing

6

05 RupaRai AuthenticatingAnaerobes–UseofMALDI-TOFMStoidentifyanaerobesintheNCTCCollection

7

06 MariaCarlosMartinsSurvivalstrategiesofClostridiumdifficiletofluctuatingconcentrationsofoxygen

8

07FilipeFolgosa Theroleofmetalloenzymesforthesurvivalof

theanaerobeClostridiumdifficileduringinfection

9

08 LesleyHoyles DiversityoftheclassCoriobacteriiawithindifferentecosystems

10

09AndrewMcDowell MultiplexPCRphylotypingofPropionibacterium

(Cutibacterium)acnes:characterisationofPCRnegativeanduntypableisolates.

11

10CarmenMcLeod PooandPuns:TherepresentationofFaecal

MicrobiotaTransplantsinEnglish-languageprintmedia(2003–2017)

12

11

AlidaVeloo ConjugativetransposonsandothermobilegeneticelementsinhumanclinicalPrevotellabiviastrains.Howmulti-drugresistancestrainsarecreated.

13

12

MichaelPerry TheSensitivityofPCRcombinedwiththeSpecificityofToxinEnzymeImmunoassay:CouldanUltra-sensitiveSingleMoleculeCountingTechnologyOfferaStandaloneSolutionforDiagnosisofClostridioidesdifficileInfection?

14

13

MichaelPerry UsingPCRinplaceofGDHasthefirstlineassayinatwo-stepCDItestingalgorithm–evidenceofhelpnothindrancefromsamplesprocessedatclinicalmicrobiologylaboratoriesinWales

15

14 MiriamCordovana EvaluationofMICROANAUT-SAnaerobesMIC

2

brothmicrodilutionpanelsforantibioticsusceptibilitytestingofanaerobes

16

15JulieWilson Descriptiveepidemiologicalanalysisof

antimicrobialresistanceinstrictanaerobesinScotland,2013-2017

17

16AndrewMcDowell Propionibacterium(Cutibacterium)acnes

infectionoftheprostateglandasarisk-factorandbiomarkerofprostatecancer?

18

17 ConorMcGrath InvestigatingGutMicrobiota-HostInteractionsinaMicroaerobicEnvironment

19

18SarahCopsey-Mawer

Fastidiousanaerobeagar(FAA)asasuitablemediumforantimicrobialsusceptibilitytesting(AST)ofanaerobesbyagardilution

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19 JackHassall EngineeringasyntheticgutmodeltoexploreClostridioidesdifficileinfections

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PosterNumber:01AnationwideretrospectivestudyofinvasiveinfectionswithFusobacteriumnecrophoruminSweden2010-2017

Background:

Fusobacteriumnecrophorumprimarilycausesthroatinfections,mainlyamongteenagersandyoungadults.Invasiveinfections,includingLemierre´ssyndrome,arerarebutafewstudieshaveshownapotentialincrease.Togainfurtherknowledgeonincidenceandclinicalpresentation,weperformedanationwidestudyinSweden.Methods:Datafrom2010-2017onbloodculturesandculturesorPCRfromsterilesitespositiveforF.necrophorumwereacquiredfromall23microbiologicallaboratoriesinSweden.Medicalrecordswerereviewed.Incidence,seasonality,ageandgenderdistribution,clinicalpresentation,careandoutcomeswereanalyzed.Results:From2010-2017300casesofinvasiveinfectionwithF.necrophorumwerediagnosedinSweden.Casesincreasedfrom2.9to5.0permillionperyearin2010-2013vs.2014-2017(p50years.Patientswithheadandneckinfectionwereyoungerthanpatientswithotherfoci(p

4

PosterNumber:02Improvingthesurveillanceofantimicrobialresistancetrendsamongstanaerobicoralpathogens

Background:

Orofacialinfectionswhentreatedbydentalpractitionersarenotroutinelysampledforcultureandsusceptibilitytesting.Consequently,antimicrobialresistantratesamongstthecausativebacteriaareunknown,hinderingeffortstoappropriatelymanagetheseinfectionsandundertakesurveillanceofresistantrates.AspartoftheCardiffhealthboardleadershipinpatientsafetyqualityimprovementprogrammeamultidisciplinarygroupwastaskedwiththeaimofimprovingsamplingpractices.

Methods:

Dentalprofessionals’limitedknowledgeregardingtheroleofdiagnosticsupportandanassociatedlackofconfidenceinsamplingprocedureswasaddressedbythecreationanddistributionoftrainingaids.SamplingandtransportationkitsweredevelopedandtransportroutesidentifiedenablingdentistswithinthecommunitysettingtosendsamplestothespecialistmicrobiologyservicesbasedatCardiffUniversityDentalHospital[UDH].AllsignificantanaerobeswereidentifiedandantibioticsusceptibilitytestingundertakenviaagardilutionthroughreferralofisolatestotheUKAnaerobicReferenceUnit.

Results:

A10%increasewasseenintheproportionofpusaspiratesbeingreceivedincomparisontopusswabs.Forthefirsttime,pusaspiratesampleshavebeenreceivedfromcommunitydentalclinics.Avastdiversityofanaerobspeciesandsusceptibilityprofileshavebeenidentified.IncreasingratesofresistanceamongstPrevotellaspeciestoamoxicillinandclindamycinisofconcern.

Conclusion:

Thisprojecthasshownthatthrougheducationalinterventionsandsupportthebarrierstothesamplingoforofacialinfectionscanbeovercome.TheultimateaimisforalldentistsinWalestohaveaccesstodiagnosticsupport.

DafyddThomas1,SelinaScotford2,JamesGillespie3,JamesCrossland3,GraceKelly4,TreforMorris2,MichaelLewis4,MelanieWilson41OralPathology,UniversityDentalHospital,Cardiff&ValeUniversityHealthBoard,Cardiff,UnitedKingdom.2UKAnaerobeReferenceUnit,PublicHealthWales,Cardiff,UnitedKingdom.3CommunityDentalService,Cardiff&ValeUniversityHealthBoard,Cardiff,UnitedKingdom.4SchoolofDentistry,CardiffUniversity,Cardiff,UnitedKingdom

5

PosterNumber:03Prevalenceofresistancegenesamonglessknowngram-negativeanaerobicbacteria,isolatedfromclinicalhumaninfections

Thepastyearstherehasbeenanincreaseinantibioticresistanceamonganaerobicbacteria.Amongtheseanaerobes,thebestknownandmoststudiedgroupistheBacteroidesgroup.OtherwellstudiedgeneraareClostridiumspp.andPrevotellaspp.Otherlessknowngeneraarenotstudied.Therefore,lackofinformationexistsabouttheirantimicrobialsusceptibilityprofileandthepresenceofresistancegenes.Inthisstudyweaimtoassesstheprevalenceofknownresistancegenesintheselesscommonspecies.

Fromtheyears2015-2017anumberofanaerobicgram-negativerods,consecutivelyisolated,werestudied.AllisolateswereidentifiedusingMALDI-TOFMS(BrukerDaltonics,Bremen,Germany).MICvaluesweredeterminedusingE-test(Biomerieux,Marcy-l’Étoile,France)andinterpretedaccordingtotheEUCASTguidelines.AtargetedPCR,usingspecificprimers,willbeusedtodetectknownresistancegenes.Generathatwillbestudiedare:Alistipesspp.,Bilophilaspp.,Dialisterspp.,Fusobacteriumspp.andSutterellaspp.

Sofar,oneDialistermicroaerophilusstrainwasresistanttometronidazole,4Sutterellawadsworthensisstrainswereresistanttoclindamycinand3S.wadsworthensisstrainswereresistanttometronidazole.Noneofthesestrainsharboredanyresistancegenes.OneAlistipesfinegoldiiisolatedidharboratetQgene,butwassensitivefortetracycline.

Theresultsobtainedsofarsuggestthatanunknownresistancemechanismmightbepresentinthetestedstrains.Also,someoftheobservedresistancemightbeintrinsic.Furtherstudiesonthissubjectiswarrantedandmorestrainswillbetested.Theresultswillbepresented.

KathleenBoiten,WillemtjeBaas,PaulineBuijs,AlidaVeloo

UniversityMedicalCenterGroningen,Groningen,Netherlands

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PosterNumber:04

DiscoveringanovelresistancegeneforcarbapenemresistanceinParabacteroidestimonensisusingWholeGenomeSequencing

Upto95%ofthehumanmicrobiomeconsistsofanaerobicbacteria.However,theyarenotaswellstudiedastheaerobicbacteria.Amongallanaerobes,theBacteroidesgroupisthebestknownandmoststudied.TherehasbeenanincreaseincarbapenemresistanceinBacteroidalesstrainswithoutaknowncause.

WeisolatedaParabacteroidestimonensisstrain,relatedtoBacteroides,resistantforamoxicillin,clindamycinandmeropenem.

ThestrainwassequencedusingtheMiseq(Illumina,SanDiego,CA),followedbyadenovoassemblyusingCLCworkbenchtocreateadraftgenome.ResistancegenesweredetectedwithResFinder(https://cge.cbs.dtu.dk/services/ResFinder/).GenomeannotationwasperformedbyRAST(http://rast.nmpdr.org/).Proteinmodelingwasperformed,ongenesofinterest,usingPhyre2(http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index)and3DLigandSite(http://www.sbg.bio.ic.ac.uk/3dligandsite/).FatCat(http://fatcat.burnham.org/)wasusedtodoapairwisestructurealignmenttocompareproteinstructures.

Thedraftgenomehadasizeof6,356,323bp,presenton95contigs.UsingResfinder1resistancegenewasfound,tetQ.Apossiblenovelresistancegeneforametalloβ-lactamaserelatedtocfiAwasfoundusingRAST.TheproteinmodelshowedsimilaritieswiththemodelforcfiAandincludesseveralZn2+ions,usedbymetalloβ-lactamasetohydrolyzecarbapenems.

Ourresultsindicatethepresenceofanotyetdescribedmetalloβ-lactamase.Therearealsoindicationsthataconjugativetransposonispresent,enablinghorizontalgenetransfer.Noermgenes,encodingforclindamycinresistance,weredetectedinthedraftgenome.

KathleenBoiten,AlidaVeloo


7

PosterNumber:05

AuthenticatingAnaerobes–UseofMALDI-TOFMStoidentifyanaerobesintheNCTCCollection

TheNationalCollectionofTypeCultures(NCTC)istheworld’soldestrepositoryofmedically-relevantbacteria.NCTCcontains5,500bacterialstrains,500ofwhichareanaerobes,withthecollectionregularlyreceivingnewTypestrainsandrecentclinicalisolates.

Fastidiousanaerobesposeauniquechallengeduringtheauthenticationprocess.NCTCmustensurethatthestrainsarefreefromcontaminationandthattheorganismsurvivesthelyophilisationprocess.Species-levelidentificationofanaerobicbacterialstrainsisachievedusingacombinationofthebothMALDI-TOFMSandVITEK2instruments.

ThisstudyevaluatestheuseofMALDI-TOFMS(Bruker)andVITEK2(BioMerieux)toidentifytheanaerobicstrainsintheNCTCcollection.176NCTCstrainsweretestedontheMALDI-TOFplatformand60strainswereidentifiedusingVITEK2.

MALDI-TOFwasabletoidentify79%anaerobestogenus-leveland64%tospecies-level.IncomparisontheVITEK2identified68%togenusand46%tospecies-level.Themainlimitingfactorforboththeseplatformsisthedatabase.ThismaybeduetonovelanaerobespeciesNCTCreceivesnotbeingpresentonthedatabases.Intheeventofnoidentification,16SribosomalRNAsequencingisemployed.Furthermore,detectionofspecificcharacteristicsiscarriedoutbyspecialistreferencelaboratoriesinCardiffandColindale,ensuringarobustcollectionofanaerobicbacteriaforuseinresearchandascontrolstrains.

HannahMcGregor,DipaliPindoria,RupaRai,SarahAlexander

NationalCollectionofTypeCultures,PublicHealthEngland,Colindale,UnitedKingdom

8

PosterNumber:06

SurvivalstrategiesofClostridiumdifficiletofluctuatingconcentrationsofoxygen

Oxygenandreactiveoxygenspecies(ROS)canreactwithmultiplecellularcomponents,leadingtotheinactivationofaplethoraofmetabolicpathways.Therefore,organismshavesystemstosenseandeliminateO2andROS,contributingtotheirsurvivalintheadversehostenvironment.

ClostridiumdifficileP28isananaerobicpathogenicbacteriumthatcontainsinitsgenomeaflavodiironprotein(FDP)andarubrerythrin(Rbr),thatareputativelyinvolvedinthedetoxificationpathwaysusedbythisorganism.

Flavodiironproteinsarewidespreadinalllifedomains,withacrucialroleinO2detoxification,throughitsreductiondirectlytowater.FDPsarecytoplasmicenzymeswithaminimalstructuralunitcomposedbytwomaindomains,ametallo-β-lactamasedomain,containingthecatalyticdiironsite,andaflavodoxindomainhavingaflavinmononucleotide.RubrerythrinsaregenerallyconsideredtoactasNADH-linkedhydrogenperoxidereductases,thuseliminatingthisROS,andarecomposedbytwoironsites:adiironcenterandarubredoxin-likeFeCys4center.

Inthisworkwithcharacterizedbiochemically,spectroscopically,structurallyandkineticallytheFDPandRbrandtheirtwoputativeredoxpartners,aHighMolecularWeightRubredoxin(HRb)andaRubredoxin(Rd).WeconfirmedtheexistenceofdirectelectrontransferbetweenHRb,RdandFDPandalsobetweenHRb,RdandRbr.Inaddition,wealsoestablishedthereactionratesforthereduction,byFDP,ofO2(0.43s-1)andH2O2(0.06s-1)andforthereductionofH2O2byRbr(1.53s-1).TheenzymaticactivityofRbrtowardsO2wasalsoinvestigated.

Romão,C.V.,etal,2016.JBIC.,21:39-52.

Martins,M.C.,etal.,2019.FRBM,inpress.

MariaCarlosMartins,BrunoSalgueiro,FilipeFolgosa,CéliaRomão,CarlosFrazão,MiguelTeixeira

ITQB-InstitutodeTecnologiaQuímicaeBiológicaAntónioXavier,Oeiras,Portugal

9

PosterNumber:07TheroleofmetalloenzymesforthesurvivaloftheanaerobeClostridiumdifficileduringinfection

Clostridiumdifficileisthemostprevalentpathogenamongallhealthcare-associatedinfections.Thisanaerobicbacteriumcancolonizethehumangut,typicallyfollowingagentsthatdisruptthenormalgutmicrobiota,likeantibiotics.Inthegut,C.difficileissubjectedtooxygen,whichithastoeliminateforsurvival.Itsgenomeencodesfortwoflavodiironproteins,capabletoreduceoxygentowater.Besides,someFDPsalsoreduceNOtoN2O,animportantfeatureasaresistancemechanismtowardsthehumaninnateimmunesystem[1,2].Flavodiironenzymesareconstitutedbyaminimalcoreoftwodomains:ametallo-β-lactamase-likeone,harboringthecatalyticcenter,followedbyashort-chainflavodoxin[1,2].MorecomplexFDPsexist,withmultipleextradomainsandredoxcenters[1,2].C.difficilecontainsa“classical”FDP,andaverycomplexone,withanextrashort-typerubredoxindomainfollowedbyanNADH:rubredoxinoxidoreductase-likeone[3].Thebiochemical,redoxandspectroscopicstudiesdemonstratedthatthisenzymereceiveselectronsdirectlyfromNADH,reducingitssubstrates,precludingtheneedforextrapartners.ThisFDPisselectiveforO2(16s-1),almost10xhigherthanwithNO.Thereactivitytowardshydrogenperoxide,asanH2O2reductasewithformationofwater,withanon-negligibleturnover(2s-1)isanoveltyinthefieldofFDPs.Sitedirectedmutantsrevealedthattherubredoxin-likecenterisessentialforelectrontransferfromNADHtothecatalyticcenter.

[1]Martinsetal,FreeRad.Biol.Med.,inpress,2019

[2]Romãoetal,J.Biol.Inorg.Chem.,21,39-52,2016

[3]Folgosaetal,Sci.Rep.,8,10164,2018

FilipeFolgosa,CatarinaAlves,MariaC.Martins,MiguelTeixeira

ITQB-NOVA,Oeiras,Portugal

10

PosterNumber:08*Flashposterpresentation

DiversityoftheclassCoriobacteriiawithindifferentecosystems

MembersoftheclassCoriobacteriiaarelittlestudied,importantfastidiousanaerobeswithinthehumangutmicrobiota.Collinsellaaerofaciensisacorememberofthegutmicrobiotathatcanpresentseveraldifferentfermentationprofileswithinindividuals,whileEggerthellalentaisimplicatedinxenobioticmetabolism.Recent‘culturomics’studieshavegreatlyincreasedthenumberofCoriobacteriiarecoveredfromhuman-associatedsamples,withthenamesofninenovelgeneracomprising12speciespublishedtodate.However,theecologicalrangeandgenomicdiversityoftheclassCoriobacteriiaarepoorlyunderstood.TaxonomicassignmentswithintheclassCoriobacteriiaareunclear,limitingthewaysinwhichdatafrom16SrRNAgene-baseddiversitystudiesinhumansandrodentmodelsareinterpreted.

Whole-genomeand16SrRNAgenesequencesofmembersoftheclassCoriobacteriiawereanalysedandassignedtothefamiliesAtopobiaceae,CoriobacteriaceaeandEggerthellaceae.Inconsistenciesbetween16SrRNAgenesequenceandwholegenomesequencedatadepositedinpublicdatabaseswereidentified.Newlyannotateddatasearchedagainst85,00016SrRNAgenesequencedatasetsincludedintheIMNGS(IntegratedMicrobialNextGenerationSequencing)databasedemonstrateshumansandrodentsharbourdistinctCoriobacteriiapopulations.MembersofthefamilyCoriobacteriaceaepredominateinthehumangut,whileEggerthellaceaearemorerepresentativeoftherodentgut.MetaboliccapabilitiesofthethreefamiliesofCoriobacteriiavarygreatly,withEggerthellaceaeasaccharolyticcomparedwithCoriobacteriaceaeandAtopobiaceae.CorrectannotationofCoriobacteriiain16SrRNAgene(andbyextensionshotgunmetagenomic)datasetsisrequiredtodeterminethecontributionsoftheseincreasinglyimportantbacteriatodifferentecosystems.

LesleyHoyles

NottinghamTrentUniversity,Nottingham,UnitedKingdom

11


MultiplexPCRphylotypingofPropionibacterium(Cutibacterium)acnes:characterisationofPCRnegativeanduntypableisolates.

We previously published a touchdownmultiplex PCR for rapid species identification and phylotyping(typesIA1,IA2,IB,IC,II,III)ofPropionibacteriumacnes.OnerecentstudyusedthePCRassaytoanalyse140clinicalP.acnes isolates (identifiedbyMALDI-TOFMS),but three isolateswerenegativeandninewereuntypableduetoatypicalPCRbandingprofiles.Wehavenowcharacterisedeightoftheseisolatesbybroad-range16SrDNAsequencingandsinglelocussequencetyping(SLST).Oneisolate,whichgaveanegative multiplex PCR reaction, was not P. acnes but P. humerisii (SLST +ve). Three isolates, whichgeneratedonlya singlePCRband (patternG)due to reactionwithmultiplexprimers (PArA-1/PArA-2)that target the16S rRNAgeneofP.acnes,were identifiedas thenewlydescribedandclosely relatedsisterspeciesP.namnetense(SLST–ve).OnefurtherisolatewithpatternGwasidentifiedasatypeIA1strain (SLST genotypeD1). Three isolateswhich gave a novel three banded profile (patternH)whereconfirmedastypeIIstrains(SLSTgenotypesK1andK2).ThesefourP.acnesstrainsarenowundergoingwholegenomesequencingtodeterminethemolecular/phylogeneticbasisoftheiratypicalPCRprofiles.In conclusion, this study has found thatMALDI-TOFMSwith VITEKMS v2.0 does not differentiateP.humerisii and P. namnetense from P. acnes. Negative or pattern G multiplex reactions with isolatesidentified as P. acnes by MALDI-TOF are suggestive of P. humerisii and P. namnetense, respectivelypendingconfirmationbySLSTand16SrDNAsequencing.

AndrewMcDowell1,JosephMcLaughlin1,Carey-AnnBurnham2,IstvanNagy31NorthernIrelandCentreforStratifiedMedicine,SchoolofBiomedicalSciences,UlsterUniversity,Londonderry,UnitedKingdom.2DepartmentofPathology&Immunology,DivisionofLaboratoryandGenomicMedicine,WashingtonUniversitySchoolofMedicine,StLouis,USA.3InstituteofBiochemistry,BiologicalResearchCentre,HungarianAcademyofSciences,Szeged,Hungary

12


PooandPuns:TherepresentationofFaecalMicrobiotaTransplantsinEnglish-languageprintmedia(2003–2017)

ThisstudyinvestigateshowEnglish-languagenewssourcesrepresentedfaecalmicrobiotatransplants(FMT)between2003and2017.Inthecontextofthisstudy,FMTisunderstoodtobetheprocessoftransferringstoolfromahealthydonortoarecipientwithadysfunctionalintestinalflorainordertorepopulatetheirgutmicrobiome.AcorpusofnewsarticlesonFMT,wasproducedbysearchingfor‘fa(e)calmicrobial’,‘microbiotatransplant’and‘stooltransplant’ontheNexis®UKnewsdatabase,generatingacorpussuitableforqualitativeanalysis(n=504articles).Inordertouncoveremergingsocialrepresentations,weinvestigatedpresscoverageofstooltransplants,aswellasbroaderthemesassociatedwithhealthandthegutmicrobiome.Ourfindingsshowthatprintmediafocusedparticularlyoncreatingnovel,mainlyhopeful,socialrepresentationsoffaecesthroughwordplayandpunning,side-liningissuesofriskandfear.Wealsoidentifychangingmetaphoricalframingsofmicrobesandbacteriafrom‘enemies’to‘friends’.Additionally,wefoundreadersarefamiliarisedwithFMTthroughthedepictionoftheprocessasbeingbothmundaneandhighlymedicalised.WeargueemergingmediarepresentationshavethepotentialtoshapemorepositivesocialrepresentationsofFMTinthegeneralpopulation,pavingthewayforFMTtobecomeamoresociallyacceptableandeffectivemedicalprocedure.Futureresearchcanbuildonthisbaselineinordertostudyhowsocialrepresentationscirculateinthewidermediaandpublicsphere,andhowtheymaychangeovertimeanddifferbetweencountriesasresearchintoFMTprogresses.

CarmenMcLeod,BrigitteNerlich

UniversityofNottingham,Nottingham,UnitedKingdom

13


ConjugativetransposonsandothermobilegeneticelementsinhumanclinicalPrevotellabiviastrains.Howmulti-drugresistancestrainsarecreated

ThebestknownanaerobicbacteriaisolatedfromhumanclinicalspecimensarethemembersoftheBacteroidesgroup.TheybelongtotheBacteroidetesphylumandcanharboraconjugativetransposon(CTn),whichtransfersbetweenstrainsinthepresenceoflowconcentrationsoftetracycline.ThebeststudiedCTnisCTnDOT,whichwasencounteredinBacteroidesthetaiotaomicron.BesidestheCTnalsothetransferofothermobilegeneticelements(MGEs)presentinthechromosomeistriggered.Inthisstudy,weassessedthepresenceofCTnsinPrevotellabiviastrains,whichbelongtothesamephylumasBacteroides.

AdraftgenomeoffiveP.biviastrains,selectedontheirantibioticsusceptibilityprofile,wasobtainedusingIlluminasequencing.AdenovoassemblywasperformedusingtheCLCGenomicsworkbench(Qiagen,Hilden,Germany).GeneswereannotatedusingRAST(ww.rast.nmpdr.org)andmanuallycheckedusingblast.Annotationandlengthofthegenewasadjustedifnecessary.

ThepresenceofaCTnwasobservedinallfivestrains.AnalysisshowedthatthefiveCTnscouldbedividedinthreedifferentgroups,whichwereallrelatedtoCTnDOT.BesidestheCTnsalsootherMGEswereencountered,harboringaselectionofresistancegenes.

AsinBacteroidesstrains,P.biviastrainscanharboraCTnrelatedtotheCTnsencounteredinBacteroides.WewillpresentwhatcouldhappenifthetransferoftheCTnintheseP.biviastrainsistriggered.

AlidaVeloo,HeinrichWinter,JohnRossen


14


TheSensitivityofPCRcombinedwiththeSpecificityofToxinEnzymeImmunoassay:CouldanUltra-sensitiveSingleMoleculeCountingTechnologyOfferaStandaloneSolutionforDiagnosisofClostridioidesdifficileInfection?

BackgroundClostridioidesdifficileinfection(CDI)continuestocausesignificantmorbidityandavoidablemortalityworldwide.Resultsfromanultra-sensitivetoxinimmunoassay(SinglulexClarityC.difftoxinsA/Bassay)werecomparedwiththoseofvariousotherdiagnosticandreferencemethods/algorithmsforthedetectionofC.difficile.Methods293residualclinicalstoolsamplesweretestedusingtheSingulexassay.Intotal,188samplesweretestedbyGDHand239weretestedbyPCR.AlltoxinBPCR(SerosepEntericBioC.difficileassay)positivesamples(n=168)andprospectivelytestedGDHsamples(n=97)werealsotestedusingmembrane-typetoxinEIA(MT-EIA;TechlabToxA/BQuikChekÒ).Culture(alcoholshockandBrazier’smedia;Oxoid)andribotyping(capillaryelectrophoresisusingBidetetal.primers)informationwasavailablefor205samples.ResultsThepositivepercentagreement(PPA)andnegativepercentagreement(NPA)oftheSingulexClarityC.difftoxinsA/Bassaycomparedwith:GDH;toxinEIA;PCR;GDH/toxinEIA;GDH/toxinEIA/PCR;PCR/toxinEIAandculturewere–61%&92%;97%&50%;69%&90%;100%&51%;81%&77%;96%&65%;and69%AND52%respectively.Conclusions

TheSingulexClarityC.difftoxinsA/BassayhadhighPPAcomparedtotoxinEIAandmultistepalgorithmsendingwithtoxinEIA,andhighNPAcomparedtoPCRandamultistepalgorithmendingwithPCR.TheSingulexClarityassayhasthepotentialtobeusedasastandalonetestforCDIdiagnosis;additionalclinicalstudiesarerequiredandwillsoonbeunderway.

MichaelPerry1,LeeGraham1,LaurenGilbert1,SwetaParida2,PhoebeKatzenbach3,JoseBaptista3,JohannaSandlund3,BethanAnderson1,SarahCopsey4,SelinaScotford4,TeforMorris11PublicHealthWalesMicrobiology,Cardiff,UnitedKingdom.2CardiffandValeUniversityHealthBoard,Cardiff,UnitedKingdom.3SingulexInc,Alameda,USA.4PublicHealthWalesMicrobiology,Cardiff,USA

15


UsingPCRinplaceofGDHasthefirstlineassayinatwo-stepCDItestingalgorithm–evidenceofhelpnothindrancefromsamplesprocessedatclinicalmicrobiologylaboratoriesinWales

Background:Guidancerecommendstheuseofatwo-stagealgorithmfordiagnosisofClostridioidesdifficileinfection(CDI)utilisingeitherPCRoraglutamatedehydrogenase(GDH)assayasthefirstlinescreen.ThereisongoingdebateastowhetherPCRisahindranceduetofearofover-diagnosis.Methods:BetweenDecember2017andMarch2019over65,000testsforCDIwereundertakeninWales.PCR(SerosepEntericBioC.difficileassay)wasemployedasthefirstlinetestfor36%ofsampleswiththeremaindertestedusingaGDHassay(TechlabC.diffChekTM-60).PositivesamplesweretestedusingatoxinEIA(TechlabToxA/BQuikChekÒ).Culture(alcoholshock&CCEY;Oxoid)andcapillaryelectrophoresisPCRribotypingwereundertakenattheUKAnaerobeReferenceUnit(UKARU).Results:TheproportionofsamplestestingpositiveusingPCRandGDHwas5.2%and8.3%withatoxinEIApositivepercentageof30%and25%respectively.Ofthesesamples73%(n=2543)ofGDHand80%(n=974)ofPCRpositivesampleswerereferredforcultureandribotyping.NoC.difficilewasisolatedfrom15%ofGDHand10.5%ofPCRpositivespecimens.Non-toxigenicstrainswereisolatedfrom6.6%ofGDHand0.2%ofPCRpositivesamplesandapproximatelyequalproportionsofbothGDHandPCRpositivesampleswereribotyped(60%vs.63%of2125samples).Conclusions:TherewasnoevidenceofincreasedascertainmentofCDIusingPCR.Infact,cultureandribotypingdemonstratedanimprovedspecificityforPCRthatishelpfulforaccurateCDIdiagnosis.MichaelPerry,BethanAnderson,SelinaScotford,Leegraham,LaurenGilbert,AlecBirchley,SarahCopsey,TreforMorris

PublicHealthWalesMicrobiology,Cardiff,UnitedKingdom

16


EvaluationofMICROANAUT-SAnaerobesMICbrothmicrodilutionpanelsforantibioticsusceptibilitytestingofanaerobes

Background:

Accurateandroutine-friendlymethodsforMICdeterminationofanaerobesaredemanded.Here,weevaluatetheperformanceofacommerciallyavailablemicrodilutionpanelincomparisontoagradientMICStriptest.

Methods:

N=163anaerobesclinicalisolates(60species,23genera)weretestedwiththeMICRONAUT-SAnaerobesMICpanel(MERLINDiagnostika,Germany).Thesamebacterialsuspensionwasusedforthepanelinoculum,andfortestingbyMICStrips(Liofilchem,Italy).Thepanelswereincubatedat37°Cinanaerobicconditionsfor≥24h,andreadvisually.Incaseofnobacterialgrowthforthegrowthcontrol,incubationwasprolongedto48-72h.ResultswereinterpretedaccordingtoEUCASTguidelines.Comparisonwasperformedintermsofessentialagreement,categoryagreementanderrorrates.

Results:

CategoryagreementwithMICstripsresultedoverall>95%(from95.6%to100%).Essentialagreementresulted91.1%forpiperacillin/tazobactam,and>95%foralltheotherantibiotics.Overall,n=15minorerrors,n=2majorerrorsandn=5verymajorerrorswereobserved.Fordoxycycline,tigecyclineandmoxifloxacin(forwhichnobreakpointsareavailable),MICRONAUTresultsdivergedfor≤1dilutionfoldfromMICStripsresults.Formostisolates(117/163)thepanelswerereadableafterovernightincubation,in42casesafter48h,in4casesafter72h.

Conclusions:

TheMICRONAUT-SAnaerobesMICpanelsprovedtobeareliablemicrodilutionmethodforantibioticsusceptibilitytestingofanaerobes,providingresultsconsistentwithgradientmethodology,withbothfastandslowgrowingspecies.Theease-of-handlingand-resultinterpretationmakesthismethodsuitableforroutineimplementation

MiriamCordovana,SimoneAmbretti

UniversityHospitalPoliclinicoSant'Orsola-Malpighi,Bologna,Italy

17


DescriptiveepidemiologicalanalysisofantimicrobialresistanceinstrictanaerobesinScotland,2013-2017

Background:Antimicrobialresistanceinanaerobicbacteriahasbeenrecognisedtobeincreasingglobally,withgrowingresistancetometronidazoleandcarbapenemsbeingofparticularconcern.HealthProtectionScotlandreceivesclinicalmicrobiologyresults,includingsusceptibilitydatafromallScottishdiagnosticlaboratoriesviathenationaldatabase;ElectronicCommunicationofSurveillanceinScotland(ECOSS).Methods:Datafrom2013to2017relatingtostrictanaerobesisolatedfromclinicalsampleswasextractedfromECOSS(de-duplicatedbasedona14dayepisodedefinition).Predominantspecies,sampletypesandsusceptibilitytoavarietyofantibioticswasassessed.AliteraturereviewwasconductedtocompareresistanceinScottishisolates,tothatreportedglobally.Results:ThemostcommonlyreportedorganismswereClostridiumperfringens(n=1802),Propionibacteriumacnes(n=1784)andBacterioidesfragilis(n=1750).Themostfrequentlyassociatedsampletypevariedbyspecies.Oftheclinicallyrelevantantibiotics,susceptibilitytestingwasperformedmostcommonlyformetronidazole(rangingfrom58-89%,dependingonspecies).Reportedresistancevariedbyagentandspecies.Conclusion:Nationally,limitedsusceptibilitytestingiscarriedoutforthemajorityofanaerobicbacteria.Wheretested,resistancetomostantibioticsincludingmetronidazolewasbroadlycomparabletothatreportedinthepublishedliterature,althoughcomparativelyhigherresistancetoclindamycinwasobservedforseveralspeciesincludingC.perfringens.WorkisongoinginScotlandtoimproveidentificationofanaerobesandstandardisationofsusceptibilitytestingforisolatesfromsterilesites.Itisanticipatedthatthiswillsupportmoretargetedtreatmentsforindividualpatientsandantimicrobialstewardshipprogrammes.

AdrianaZalewska,JulieWilson,EdwardMcardle,MichaelLockhart,WilliamMalcolm

HealthProtectionScotland,Glasgow,UnitedKingdom

18


Propionibacterium(Cutibacterium)acnesinfectionoftheprostateglandasarisk-factorandbiomarkerofprostatecancer?

Prostate cancer (PCa) is themost commonmale cancer in the UK, killing approximately 11,000menevery year. There is now increasing interest in the role played by the anaerobic bacteriumPropionibacteriumacnes in theaetiologyof this condition via chronic, intracellular andasymptomaticinfection of the prostate leading to oncogenesis. To investigate this,we developed a novel Taqman®qPCRassayforretrospectivedetectionofP.acnes in formalin-fixedparaffin-embeddedtissuesectionspreparedfromarchivedprostatebiopsysamples.Atotalof81biopsysampleswereexaminedfrom53patients with prostate carcinoma, versus 111 samples from 60 patients whose biopsies werehistologicallynormal.Ourassayrevealedthat35%ofmenwithPCawerepositiveforthepresenceofP.acnesineitheroneorbothprostatelobescomparedtoonly8%ofnormaltissue(Fisher’sexacttest,2-sided;p

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InvestigatingGutMicrobiota-HostInteractionsinaMicroaerobicEnvironment

Thegutmicrobiotahasanimportantroleinmaintainingintestinalhealthandprotectingagainstentericinfections(colonisationresistance).Neverthelessthemajorityoftheseinteractionshaven’tbeenexplored,largelyduetoalackofexperimentalmodelsystemsthatcancultureoxygen-sensitivecommensalsalongsideintestinalcells.Inthisproject,wehaveestablishedanovelinvitromodelsystemofthehumanintestinalepithelium(VerticalDiffusionChamber,VDC)whichalsosupportsgrowthofstrictlyanaerobicbacteria.WehaveappliedthissystemtoinvestigatetheinteractionsofgutsymbiontRuminococcusgnavuswithafunctioningmucus-producingepithelium,establishedusingT84andgoblet-likeLS174Tcelllines,anditseffectoninfectionwithfoodbornepathogenenteropathogenicE.coli(EPEC).

InitialworkfocusedonidentifyingaculturemediumthatsupportsR.gnavusandEPECgrowthwhilstmaintainingepithelialintegrityandbarrierfunction.Thishasbeenachievedbyestablishingbacterialgrowthcurvesindifferentmediaandassessingepithelialbarrierfunctionbytransepithelialelectricalresistanceandimmunofluorescencestaining(IFS)oftight-junctionproteinoccludin.FurtherIFSdemonstratedthatintroductionofLS174TcellstotheepitheliumcausesmucinsecretionandfacilitatescolonisationbyR.gnavus.Co-cultureofEPECwithR.gnavusreducesnumbersofviableandadherentEPEC,butonlywhenLS174Tarepresent.

ThesedataindicatepotentialcolonisationresistanceactivitybyR.gnavus,discoveredbyutilisingamodelsystemthatsupportsanaerobiccultureandafunctioningepitheliumside-by-side.FutureworkwillinvestigatecolonisationresistanceactivityforapanelofR.gnavusstrainsandattempttoelucidatemechanismsbehindthisactivity.

ConorMcGrath1,2,AndrewBell2,EmmanuelleCrost2,NathalieJuge2,StephanieSchüller11UEAMedicalSchool,Norwich,UnitedKingdom.2QuadramInstituteBioscience,Norwich,UnitedKingdom

20


Fastidiousanaerobeagar(FAA)asasuitablemediumforantimicrobialsusceptibilitytesting(AST)ofanaerobesbyagardilution

Background:

Agardilutionisthereferencemethodforantimicrobialsusceptibilitytesting(AST)ofanaerobes,butcurrentlytheonlyverifiedandpublishedmethodutilisessupplementedbrucellaagar,whichisnotreadilyavailableintheUK.AsFAAisoftenthemediumofchoiceforprimarycultureofanaerobesinclinicallaboratories,theaimofthisstudywastoinvestigatethesuitabilityofthismediumasanalternativetobrucellaagarforthereferencemethod.

Methods

OnehundredisolatessubmittedtotheUKARU,allwithpre-determinedMICsto:clindamycin;meropenem;metronidazole;penicillinanddoxycycline,weretested.MICswereobtainedbyagardilutionusingbothin-houseproducedFAA,supplementedwith5%defibrinatedhorseblood,andsupplementedbrucellabloodagar(BRU).EndpointswereselectedaccordingtoCLSIguidanceandinterpretedusingEUCASTclinicalbreakpointsforanaerobes(FDAguidancefordoxycycline).

Results:

ForthemajorityofisolatesthecorrelationbetweenFAAandBRUMICswasgood.Errorswerefoundforeachantimicrobial,withthehighestnumbersrecordedformetronidazoleanddoxycycline.Themajorityoferrorswerewithin1-2dilutions,butspannedtheclinicalbreakpoints.Othersweresinglereplicateerrors,whichwereanomalousagainstaminimumofthreeadditionalresults.SeveralspeciessuchasP.distasonisandC.ramosumdemonstratedraisednumbersoferrors/variableresultsandwillrequirefurtherinvestigation.

Conclusions:

OverallthereproducibilityofagardilutiontestingonFAAcomparedtoBRUwasgood,suggestingthatFAAisasuitablemediaforASTofanaerobes.Furtherassessmentofseveralspeciesisrequired.

SarahCopsey-Mawer,SelinaScotford,BethanAnderson,CarolDavis,MichaelPerry,TreforMorris,HarrietHughes

UKAnaerobeReferenceUnit,PublicHealthWales,Cardiff,UnitedKingdom

21


EngineeringasyntheticgutmodeltoexploreClostridioidesdifficileinfections

Our intestines play home to over one thousand bacterial species, often referred to as the gutmicrobiota.Thismicrobiotaprofoundlyaffectsourbodies,providinganarrayofbenefits.Disturbancestothiscommunityareassociatedwithpathogenicinfectionanddiseasessuchasobesity,diabetesandinflammatory bowel disease.Our aim is to engineer a synthetic microbiota, with an emphasis ontrackingindividualspecies.Introducingthiscommunityintoourinvitrogutepithelialculturesystemwillprovideinsightsintopathogeninfectionstrategies,andmechanismsbywhichthemicrobiotageneratesresistance.Thistoolcanbeusedforthescreeningofdrug/probioticcandidates,potentiallyreducingtheneed for animals. UsingPMA-qPCR we are able to track up to ten single species in a mixedbiofilm.Clostridium difficilean opportunistic pathogen targets the gut during times of depletedmicrobiota.WehavebegunusingourtrackingtechnologytoseehowarepresentativemicrobiotareactstoaC.difficile invasion.Alongsidethis,we investigatedtheeffectof thegutcommensal -BacteroidesdoreionC.difficileutilizingclassicalquantification.Inbothastandardbiofilmandinourinvitromodel,weseeareducednumberofC.difficilewheninmixedculture.

JackHassall,MeeraUnnikrishnan

UniversityofWarwick,Coventry,UnitedKingdom

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