Engineering6(2020)49–55Contents lists available at ScienceDirectEngineeringResearch
AnimalDiseaseResearch—Article
FirstIsolationandCharacterizationofOchrobactrumanthropifromPig
ShijiangGua,#,RuiqingHoua,#,ShengguoGaoa,ZheSuna,XiangdongLia,LufengZhaia,YunyunJina,QiaoyanZhua,YonghongLiaoa,?,KegongTiana,b,?abNationalResearchCenterforVeterinaryMedicine,Luoyang471000,ChinaCollegeofAnimalScienceandVeterinaryMedicine,HenanAgriculturalUniversity,Zhengzhou450002,Chinaarticleinfoabstract
OneGram-negativeBacilluswasisolatedfromabrainsampleofapigwithneurologicalsymptoms.Pathologicalexaminationshowedmeningitisatnecropsy.Ochrobactrumanthropi(O.anthropi)wassuc-cessfullyisolatedfromthebrainsampleandwascon?rmedbybiochemicalreactionresults(API20NE)andgenesequencing.Thestrainwashighlyresistanttob-lactamantibiotics.Micewereexperimen-tallyinfectedwithO.anthropiandshowedtypicalmeningitis.Thisisthe?rstreportonO.anthropiiso-latedfromapig,andindicatesthatO.anthropimayhaveabroaderhostspectrumofinfection.ó2020THEAUTHORS.PublishedbyElsevierLTDonbehalfofChineseAcademyofEngineeringandHigherEducationPressLimitedCompany.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).Articlehistory:Received18March2019Revised31July2019Accepted2August2019Availableonline25October2019Keywords:OchrobactrumanthropiPigbrain1.IntroductionOchrobactrumanthropi(O.anthropi)isaGram-negativeBacillusthatistremendouslyabundantintheenvironment.ItwasformerlyplacedinthecategoryofCDCgroupVd[1],whichbelongstothefamilyBrucellaceae[2].O.anthropiwaspreviouslyconsideredtobeahumanconditionalpathogenthatmainlyinfectedimmunocompromisedpatients[1,3,4].However,severalreportshaveindicatedthatO.anthropicanalsocauseinfectionsinhealthyhumans[5–7].ThemanifestationsofO.anthropiinfectionsareendophthalmitis[6],endocarditis,meningitis,osteochondritis,osteomyelitis,pancreaticabscess,puncturewound,pelvicabscess,andurinarytractinfection[8–14].Besideshumanbeings,O.anthropihasbeenreportedtoinfectothermammals.Francietal.[15]werethe?rsttoreportahealthycaninebeinginfectedwithO.anthropi;thisoccurredafterroutinedentalcleaningasaresultoftheanestheticsolutionbeingcontaminatedwithO.anthropi.El-Adawyetal.[16]separatedO.anthropifromthececumofatur-key.Todate,O.anthropihasnotbeenreportedasoccurringinanyotherspecies.Inthisstudy,weisolatedandcharacterizedonestrainofO.anthropifromapigforthe?rsttime.2.Materialsandmethods2.1.IsolationofbacteriafrompigclinicalsamplesThepigbrainsamplewasobtainedfromacommercialfarminJiangxiProvince,China.Thepighadneurologicalsymptoms,andthepathologicalexaminationshowedmeningitisatnecropsy.Histopathologicalexaminationandbacterialseparationwerecar-riedoutfromthebrainsamples.Swabsweretakenfromthebrainsampleunderasepticconditionstoavoidcontamination.Theiso-latewasculturedontryptonesoyagar(TSA;BDDifcoTM,Becton,DickinsonandCompany,USA)containing10%newbornbovineserum(ZhejiangTianhangBiotechnologyCo.,Ltd.,China)at37°Cfor24h.GramstainingwasconductedaccordingtothemethoddescribedbyGerhardtetal.[17].Thebiochemicalreactionwaspro?ledusingtheAPI20NEassay(bioMérieuxInc.,France),accordingtothemanufacturer’sinstruction.2.2.DNApreparationDNAfrombacterialsamplesculturedfor24hwasextractedbyaGENEWIZBacteriaGenomicDNAKit(GENEWIZ,China)accordingtothemanufacturer’sinstruction.2.3.PCR,genesequencing,andconstructionofphylogenetictrees?Correspondingauthors.#E-mailaddresses:ebrain04@163.com(Y.Liao),tiankg@263.net(K.Tian).Theseauthorscontributedequallytothiswork.
The16SrRNAgeneandrecAgenewereampli?edusingthemethodspreviouslydescribedbyScholzetal.[18].Theforward50S.Guetal./Engineering6(2020)49–55andreverseprimers27f(50-AGAGTTTGATCMTGGCTCAG-30)and1492r(50-AAGTCGTAACAAGGTARCCG-30)wereusedtogenerateanapproximately1400bpfragmentofthe16SrRNAgene.Thepro-?leofthepolymerasechainreaction(PCR)cyclewasasfollows:94°Cfor5min,30cyclesat94°Cfor60s,56°Cfor30s,72°Cfor90s,and10minat72°C.ThePCRproductswereanalyzedin1%agarosebygelelectrophoresis.TheprimersforrecAarerecA-f(50-ATGTCTCAAAATTCATTGCGAC-30)andrecA-r(50-AGCATC-TTCTTCCGGTCCGC-30);thesewereusedtoobtaina1065bpsizebandoftherecAgene.ThePCRcyclewasasfollows:94°Cfor5min,30cyclesat94°Cfor30s,58°Cfor30s,72°Cfor60s,and10minat72°C.ThePCRproductswereanalyzedbyelec-trophoresisin1%agarosegel.ThePCRproductsweresenttoGENEWIZforsequencing.ThesequenceswereBLASTed(whereBLASTstandsforBasicLocalAlignmentSearchTool)byBLASTnxintheNationalCenterforBiotechnologyInformation(NCBI)database.CLUSTALXwasused[19]toalignthesequences.Phylogenetictreeswerebuiltbasedonthe16SrRNAgene(rrs)andtherecAgeneusingtheneighbor-joiningmethodinKimura’stwo-parametermodel[20]inMEGAVersion5[21,22].Thepercentageofreplicatetreesinwhichtheassociatedtaxaclusteredtogetherinthebootstraptest(1000repli-cates)wereshownnexttothebranches[23].2.4.GenomicsequencingandBLASTThegenomicsequenceswereBLASTedbyBLASTgenomesintheNCBIdatabase.PartialsequencesoftherecAand16SrRNAandthegenomicsequencesOchrobactrumandBrucellaspp.weredepositedinGenBankwiththeaccessionnumbersdocumentedinTableS1inAppendixA.Supplementarydata.2.5.AntibioticsusceptibilityandresistancetestingTheisolatewassubjectedtoantibioticsusceptibilityandresis-tancetestingbythediskdiffusionmethodpresentedbyBaueretal.[24].TheisolatedstrainwasculturedonTSAcontaining10%new-bornbovineserumat37°Cfor24handwasthendilutedinphosphate-bufferedsaline(PBS)(0.01moláLà1,pH7.2)at1?108CFUámLà1(whereCFUstandsforcolonyformingunits).ThebacterialsuspensionwasspreadontheTSAcontaining10%newbornbovineserumwithasterilecottonswab,andthepaperantibioticdisks(HangzhouMicrobialReagentCo.,Ltd.,China)wereplacedontheinoculatedagarsurface.Theplateswereincubatedat35°Cfor24hpriortodetermination.ThezonediametersofeachdrugwereinterpretedusingthecriteriapublishedbythePerformanceStandardsforAntimicrobialDiskandDilutionSuscep-tibilityTestsforBacteriafromAnimals[25].2.6.MouseexperimentalinfectionstudyTheisolatedstrainwasculturedonTSAcontaining10%new-bornbovineserumat37°Cfor24handwasthendilutedinPBS(0.01moláLà1,pH7.2)toobtainaseriessuspensionofabout5?109–2?1010CFUámLà1.BALB/cmicewereintraperitoneallyinjectedwiththebacterialsolutionat1?109–4?109CFUpermousewithavolumeof0.2mLpermouse.Histopathologicalobservationwascarriedoutforthemousebrainsamples.Theani-maltrialfollowedtheanimalprotocolapprovedbytheAnimalCareandEthicsCommitteeoftheChinaNationalResearchCenterforVeterinaryMedicine.2.7.HistopathologicalobservationThebrainsampleswere?xedwith10%bufferedformalinovernight,followedbyembeddinginparaf?n.Braintissuesweresectionedandstainedwithhematoxylinandeosinforhistopatho-logicalevaluation.3.Results3.1.HistopathologicalexaminationandbacterialisolationThehistopathologicalresultsforthepigbrainsampleshowednecroticnodulesinthewhitematterofthebrain,necrosisofgraymatterneurons,andalargenumberofneutrophilin?ltrations,asshowninFig.1.ThebrainsamplewasstreakedontoTSAplatestoobtainpurecolonies.Thecoloniesweresmallandsmooth;thecellsinthecolonieswereshowntobeGram-negativebacillusafterGramstaining(Fig.2).Biochemicalidenti?cationrevealedthattheisolatedstrainwasoxidaseandcatalasepositive.Theisolatewasidenti?edasO.anthropi(99.9%probability)byAPI20NEtest(Table1).ThisisolatewasnamedJXLH03B.3.2.SequencingandphylogeneticanalysisThesequenceoftheJXLH03B16SrRNAgenewascompletelyidenticaltothatofOchrobactrumsp.(GenBankaccessionNo.MH201346.1),O.anthropi(GenBankaccessionNo.LT671861.1),andOchrobactrumlupini(O.lupini)(GenBankaccessionNo.Fig.1.Histopathologicalobservationofthepigbrainsample(hematoxylinandeosinstaining(H&E),20?).(a)Necroticnodulesinthewhitematterofthebrain(shownbythearrow);(b)necrosisofgraymatterneurons(shownbythetriangle)andneutrophilin?ltration(shownbythearrow).S.Guetal./Engineering6(2020)49–5551Fig.2.Colonymorphologyandcellmorphologyoftheisolate.(a)Colonymorphologyoftheisolate;(b)microscopicexaminationofcellmorphology(100?)afterGramstaining.Table1
API20NEidenti?cationoftheJXLH03Bisolate.TestnameNO3TRPGLUADHUREESCGELPNPGGLUARAMNEMANNAGMALGNTCAPADIMLTCITPACOXAPI20NEReaction/enzymeNitratesreductionIndoleformationGlucosefermentationArgininedihydrolaseproductionUreaseproductionHydrolysis(b-glucosidase)Hydrolysis(protease)b-galactosidaseGlucoseassimilationArabinoseassimilationMannoseassimilationMannitolassimilationN-acetyl-glucosamineassimilationMaltoseassimilationPotassiumgluconateassimilationCapricacidassimilationAdipicacidassimilationMalicacidassimilationTrisodiumcitrateassimilationPhenylaceticacidassimilationCytochromeoxidaseIncubationtime(h)Result+ààà+àààà+à+à+++++àà++àààà++++àà++O.anthropi99.9$48244824482448244824482448244824482448244824482448KU217325.1).TherecAgenewasalsoBLASTedandwasshowntobecompletelyidenticaltoO.anthropi(GenBankaccessionNo.LT671861.1).Phylogeneticanalysiswasperformedbasedonthe16SrRNAgene.AsshowninFig.3,JXLH03BwasgroupedwithO.anthropiLMG7991,O.anthropiOAB,O.anthropiLMG5140,O.anthropiLMG3333,O.anthropiLMG3333T,O.anthropiDSM14396,O.anthropiATCC49188,O.anthropi7b2C,andO.lupiniLMG1821.Bycontrast,JXLH03BwasgroupedwithO.anthropiDSM14396,O.anthropiLMG3333,andO.anthropi10CS0094,basedontherecAgene(Fig.4).3.3.GenomicsequencingandBLASTThegenomicsequencingresultsoftheJXLH03Bisolateweretwosequenceswithatotallengthof4725913bp.TheG+Ccon-tentwas56.01%.Thelengthsofsequence1andsequence2were52S.Guetal./Engineering6(2020)49–55Fig.3.Phylogeneticanalysisbasedonthe16SrRNAgeneusingtheCLUSTREEneighbor-joiningmethod.Scalebar:0.002divergentresiduespersite.Thesigni?canceofeachbranchisindicatedbyabootstrapvaluecalculatedfor1000subsets.2641072bpand2084841bp,respectively.TheBLASTresultsshowedthatthesimilaritybetweentheJXLH03BisolatesequencesandthetwodepositedgenomesequencesofO.anthropiwas97.54%–98.08%(Table2).3.5.AntibioticsensitivitytestAntimicrobialsusceptibilitytestingwasperformedusingadiskdiffusiontestfollowingtheguidelinesofthePerformanceStan-dardsforAntimicrobialDiskandDilutionSusceptibilityTestsforBacteriafromAnimals[25].TheJXLH03Bisolatewassusceptibletogentamicinsulfate,amikacin,enro?oxacin,cipro?oxacin,doxy-cycline,and?orfenicol,butwasresistanttopenicillinsodium,cef-tiofur,kanamycinsulfate,lincomycin,andtylosin(Table4).3.4.MouseexperimentalinfectionstudyBALB/cmicewereinfectedwithJXLH03Bat1?109–4?109CFUpermouse.Mousemortalityrangedfrom0/5to5/5(Table3).Thebrainsofallthedeadmiceweresampledforhistopathologicalexamination.Allofthemicethatdiedafterinfec-tionhadin?ammationinthebraintissue.Thehistopathologicalresultsshowedseveralnecroticfociinthebrain.Thebrainparenchymawaslique?ed,andalargeamountofneutrophilsnecrosisaggregatedtoformnodules(shownbythearrowinFig.5).Someglialcellswerefoundtocollapse(Fig.5).4.DiscussionO.anthropiisanaerobic,non-lactose-fermenting,motile,oxidase-positive,urease-positive,Gram-negativeBacillusprevi-ouslyknownas‘‘AchromobactergroupVd”[1].Thebiochemicalreactionpro?les(API20NE)identi?edtheJXLH03BstrainasO.anthropi.ThegenusOchrobactrumbelongstothefamilyS.Guetal./Engineering6(2020)49–5553Fig.4.PhylogeneticanalysisbasedontherecAgeneusingtheCLUSTREEneighbor-joiningmethod.Scalebar:0.01divergentresiduespersite.Thesigni?canceofeachbranchisindicatedbyabootstrapvaluecalculatedfor1000subsets.Table2
SequencesimilarityofgenomicsequencesbetweenJXLH03BandtwodifferentO.anthropistrains.O.anthropistrainChromosomeGenBankNumberSequencesimilarityJXLH03Bsequence1ATCC49188OABChromosomeChromosomeChromosomeChromosome1212CP000758.1CP000759.1CP008820.1CP008819.198.08%—98.08%—JXLH03Bsequence2—97.82%—97.54%BrucellaceaeandhasaclosephylogeneticrelatednesstoBrucella[26];thegenusOchrobactrumcomprisesabout16species[27].16SrRNAgeneanalysiswasusedfortheidenti?cationanddiffer-entiationofOchrobactrumandBrucellaspp.[2,26,27].However,thisapproach—particularlypartialsequencing—ispronetomisidentifyingthesepathogensbecauseoftheirhighsequencesimilarities[27].Meanwhile,recAanalysisprovidesmoreaccurateidenti?cationanddifferentiation[18,27].Weperformedthe16SRNAgenesequencingalignmentoftheJXLH03Bisolate,whichcouldnotbecompletelyidenti?edasO.anthropi;however,theresultoftherecAgenesequencingcomparisonidenti?edtheJXLH03BisolateasO.anthropi.Theinfectiontestinmiceshowedthatthestrainhadacertainpathogenicitythatledtobrainlesions.TotestthepathogenicityoftheJXLH03Bstrainonpigs,nine80-day-oldhealthypigswerechallengedbytrachealinjectionatdosesof1.2?109,1.2?1010,
首次从猪体内分离鉴定人苍白杆菌 - 图文
