Microbiological and bioinformatics analysis of primary Sj?gren’s syndrome patients with normal salivation§

摘要

Background:ReducedsalivationisconsideredamajorclinicalfeatureofmostbutnotallcasesofprimarySj?gren’ssyndrome(pSS).Reducedsalivaflowmayleadtochangesinthesalivarymicrobiota.Thesechangeshavemainlybeenstudiedwithculturethattypicallyrecoversonly65%ofthebacteriapresent.Objective:Thisstudywastousehighthroughputsequencing,coveringbothcultivatedandnot-yet-cultivatedbacteria,toassessthebacterialmicrobiotaofwholesalivainpSSpatientswithnormalsalivation.Methods:BacteriaofwholeunstimulatedsalivafromninepSSpatientswithnormalsalivationflowandfromninehealthycontrolswereexaminedbyhighthroughputsequencingofthehypervariableregionV1V2of16SrRNAusingthe454GSJuniorsystem.Rawsequencereadsweresubjectedtoaspecies-level,reference-basedtaxonomyassignmentpipelinespeciallydesignedforstudyingthehumanoralmicrobialcommunity.EachofthesequencereadswasBLASTN-searchedagainstadatabaseconsistingofreferencesequencesrepresenting1,156oraland12,013non-oralspecies.Unassignedreadswerethenscreenedforhigh-qualitynon-chimerasandsubjectedtodenovospecies-leveloperationaltaxonomyunit(OTU)callingforpotentialnovelspecies.Downstreamanalyses,includingalphaandbetadiversities,wereanalyzedusingtheQuantitativeInsightsintoMicrobialEcology(QIIME)pipeline.TorevealsignificantdifferencesbetweenthemicrobiotaofcontrolsalivaandSj?gren’ssaliva,astatisticalmethodintroducedinMetastatswww.metastats.cbcb.umd.eduwasused.Results:SalivaofpSSpatientswithnormalsalivationhadasignificantlyhigherfrequencyofFirmicutescomparedwithcontrols(p=0.004).Twoothermajorphyla,SynergistetesandSpirochaetes,weresignificantlydepletedinpSS(p=0.001forboth).Inaddition,wesawanearly17%decreaseinthenumberofgenerainpSS(25vs.30).WhilePrevotellawasalmostequallyabundantinbothgroups(25%inpSSand22%incontrols),aboutatwofoldincreaseinpSSofStreptococcus(28%vs.17%)andVeillonella(26%vs.12%)wasdetected.Prevotellamelaninogenicawasthemajorspeciesincontrols(13%)whileVeillonellaatypicaandtheVeillonellaparvulagroupsdominatedinpatientsamples(14and14%).ThescarcityinbacterialspeciesinpSScomparedwithcontrolswasalsodemonstratedbyalphaandbetadiversityanalyses,aswellasreadabundancedepictedinaphylogenetictree.Conclusions:WhileFirmicuteswassignificantlyhigherinpSSpatientsthanincontrols,SynergistetesandSpirochaetesweresignificantlylower.Thenumberofbacterialgeneraandspecieswasalsolower.ThesedatashowedthatmicrobialdysbiosisisanotherkeycharacteristicofpSSwholesalivawhichcanoccurindependentofhyposalivation.Keywords:primarySj?gren’ssyndrome;wholesaliva;normalsalivaryflowrate;bacteria;highthroughputsequencing;pyrosequencing(Published:20October2016)Citation:JournalofOralMicrobiology2016,8:31119-http://dx.doi.org/10.3402/jom.v8.31119