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@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
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02DafyddThomas Improvingthesurveillanceofantimicrobial
resistancetrendsamongstanaerobicoralpathogens
4
03KathleenBoiten Prevalenceofresistancegenesamongless
knowngram-negativeanaerobicbacteria,isolatedfromclinicalhumaninfections
5
04KathleenBoiten Discoveringanovelresistancegenefor
carbapenemresistanceinParabacteroidestimonensisusingWholeGenomeSequencing
6
05 RupaRai AuthenticatingAnaerobes–UseofMALDI-TOFMStoidentifyanaerobesintheNCTCCollection
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06 MariaCarlosMartinsSurvivalstrategiesofClostridiumdifficiletofluctuatingconcentrationsofoxygen
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07FilipeFolgosa Theroleofmetalloenzymesforthesurvivalof
theanaerobeClostridiumdifficileduringinfection
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08 LesleyHoyles DiversityoftheclassCoriobacteriiawithindifferentecosystems
10
09AndrewMcDowell MultiplexPCRphylotypingofPropionibacterium
(Cutibacterium)acnes:characterisationofPCRnegativeanduntypableisolates.
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10CarmenMcLeod PooandPuns:TherepresentationofFaecal
MicrobiotaTransplantsinEnglish-languageprintmedia(2003–2017)
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11
AlidaVeloo ConjugativetransposonsandothermobilegeneticelementsinhumanclinicalPrevotellabiviastrains.Howmulti-drugresistancestrainsarecreated.
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MichaelPerry TheSensitivityofPCRcombinedwiththeSpecificityofToxinEnzymeImmunoassay:CouldanUltra-sensitiveSingleMoleculeCountingTechnologyOfferaStandaloneSolutionforDiagnosisofClostridioidesdifficileInfection?
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MichaelPerry UsingPCRinplaceofGDHasthefirstlineassayinatwo-stepCDItestingalgorithm–evidenceofhelpnothindrancefromsamplesprocessedatclinicalmicrobiologylaboratoriesinWales
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14 MiriamCordovana EvaluationofMICROANAUT-SAnaerobesMIC
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brothmicrodilutionpanelsforantibioticsusceptibilitytestingofanaerobes
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15JulieWilson Descriptiveepidemiologicalanalysisof
antimicrobialresistanceinstrictanaerobesinScotland,2013-2017
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16AndrewMcDowell Propionibacterium(Cutibacterium)acnes
infectionoftheprostateglandasarisk-factorandbiomarkerofprostatecancer?
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17 ConorMcGrath InvestigatingGutMicrobiota-HostInteractionsinaMicroaerobicEnvironment
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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
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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
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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
UniversityMedicalCenterGroningen,Groningen,Netherlands
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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
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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
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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
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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
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PosterNumber:09*Flashposterpresentation
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
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PosterNumber:10*Flashposterpresentation
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
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PosterNumber:11*Flashposterpresentation
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
UniversityMedicalCenterGroningen,Groningen,Netherlands
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14
PosterNumber:12*Flashposterpresentation
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
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PosterNumber:13*Flashposterpresentation
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
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16
PosterNumber:14*Flashposterpresentation
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
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17
PosterNumber:15*Flashposterpresentation
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
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18
PosterNumber:16*Flashposterpresentation
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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PosterNumber:17*Flashposterpresentation
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
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20
PosterNumber:18*Flashposterpresentation
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
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21
PosterNumber:19*Flashposterpresentation
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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