微生物抗药性及生态效应

微生物抗药性及生态效应第一页，共83页。微生物抗药机制基因水平转移环境中的抗性基因第二页，共83页。MycobacteriumtuberculosisEnterococcusfaecalisStreptococcuspneumoniaeE.coli第三页，共83页。第四页，共83页。抗生素抗药性已经成为全球性的重大问题

Resistance1998-2004-%%Resistance1998-2004-%StreptococcuspneumoniaeinUSA第五页，共83页。第六页，共83页。S.

aureusPenicillin[1950s]Penicillin-resistantS.aureusEvolutionofDrugResistanceinS.aureusMethicillin[1970s]Methicillin-resistant

S.aureus(MRSA)Vancomycin-resistantenterococci(VRE)Vancomycin[1990s][1997]Vancomycinintermediate-resistantS.aureus

(VISA)

[2002]Vancomycin-resistantS.aureus第七页，共83页。EscherichiacoliinAustralia201011KlebsiellapneumoniaeIndia200912Kpneumoniae，EcoliIndia,Pakistan,andtheUK20108CitrobacterfreundiiFrench

returningfromIndia201010屎肠球菌，鲍曼不动杆菌201010超级细菌（Superbug）NDM-1NewMetallo--LactamaseNDM-1超级细菌在世界各地被发现。第八页，共83页。我国发现NDM-1超级细菌第九页，共83页。第十页，共83页。超级细菌对多种抗生素均有抗性第十一页，共83页。Tigecycline

新批准的新型抗生素

[简介]：Tigecycline(商品名为Tygacil；惠氏公司生产)2005年6月获得FDA批准，用于治疗细菌感染。这是一种称为glycylcyclines的新型抗生素第一个获得批准的产品。该类抗生素用于克服早期四环素类抗生素的耐药性。除了Tigecycline，在过去的40年里，只有两个新型抗生素上市，分别是辉瑞公司的linezolid(Zyvox)和Cubist公司的daptomycin(Cubicin)。

多粘菌素Colistin(polymyxinE)是是混合物多聚氨基酸，主要针对革兰氏阴性杆菌。第十二页，共83页。第十三页，共83页。超级细菌（Superbug）Plasmids

Chennai50kbto350kb；Haryana118kb(54%)or50kb(36%)；UKisolates80kbtogreaterthan500kb.MostblaNDM-1positiveplasmidswerereadilytransferableandpronetorearrangement,losingor(morerarely)gainingDNAontransfer.质粒的可移动性和可塑性可能意味着在细菌中的广泛传播和不断变异。第十四页，共83页。加拿大魁北克省舍布鲁克大学附属医院的专家日前透露，从2003年年初至今，一种普通的肠道细菌——梭状芽孢杆菌历经两年变异，已成为致命的“超级病菌”，它可引起65岁以上老年人和服用抗生素的病人产生严重痢疾，并最终致死。该病菌已使这家医院的100名病人死亡，如不采取紧急行动，这种“超级病菌”很可能会引发一场致命传染病的蔓延。

第十五页，共83页。2010年9月5日，日本媒体近日披露，有46名患者在日本帝京大学医学部附属医院住院期间感染了几乎可以抵抗所有抗生素的超级细菌——多重耐药鲍曼不动杆菌，其中9名患者的死亡因感染此种病菌所致。该医院曾刻意隐瞒感染事件，导致日本首次出现大规模不动杆菌感染，日本警方遂决定传唤该院医生，展开调查。经检测发现，患者感染的不动杆菌能抵抗多种抗生素，目前只有先锋霉素族的一部分抗生素对这一病菌有效。

第十六页，共83页。-MRSAinfectionofhipscrew-VnMIC<1mg/L→Vntreatment-d55,developedEnterobacterinfection -ciproaddedtoVnfor6weeks-d99,VnMIC=4mg/L -d159,Vndiscontinued-d174,readmittedtohospital,Vntreatment -VnMIC=4mg/L-d188,linezolid+rifampin-d198,infectioncleared-12surgeriesincludingremovalofinfectedboneAcaseofresistantSaureusinpatientCanada-Edmonton-Websteretal.,2007第十七页，共83页。细菌耐药机制DrugImpermeabilityDrugEffluxTargetmodificationDrugInactivationOnetypeofantibioticcanbeovercomebydifferentmechanismsDifferentclassesofantibioticscanhavethesamemechanismsofresistance第十八页，共83页。孔蛋白改变，细胞壁/膜通透性改变万古霉素（vancomycin）：由一种链霉菌产生的、结构复杂的糖肽类抗生素，专一地抑制肽聚糖的生物合成。万古霉素通过抑制细菌的生长和繁殖来杀死细菌。这种药物通过干扰细菌细胞壁结构中的一种关键组分来干扰细胞壁的合成，抑制细胞壁中磷脂和多肽的生成，只是针对革兰氏阳性菌，对厌氧菌和革兰氏阴性细菌无效。万古霉素的药力较强，且有肾毒性，在其他抗生素对病菌无效时才会被使用，也就是所谓的最后一线药物。

第十九页，共83页。Howdenetal.2006VISAstrainshavethickercellwalls第二十页，共83页。-adsorptionofVnbycell-wallD-ala-D-alaresiduesincreased,diffusiondecreased-drugispreventedfromreachingitsactivesiteVnsensitiveVnresistantVISAstrainshavethickercellwalls第二十一页，共83页。TheCaseofCMRSA-1MRSAVISA(4mg/L)potentialhVISA(<4mg/L)第二十二页，共83页。TheCaseofCMRSA-1敲除株野生型耐药株45.7+/-6.722.1+/-2.529.0+/-3.2JolyneDrummelsmith,JournalofProteomeResearch2007,6,4690–4702第二十三页，共83页。主动外排泵因为外排泵的主动输出抗生素，因而细菌内达不到治疗浓度。一些泵具有相对较强的特异性，如四环素运输泵，而其他输出泵具有广泛的底物选择性和耐药性。细菌体内具有大量的输出泵，用于运输代谢物和外来有毒物质。第二十四页，共83页。Linezolid-oxazolidinone第二十五页，共83页。SusceptibilitylevelsofS.pneumoniaeisolates

LZD,linezolid;CHL,chloramphenicol;FFC,florfenicol;ERY,erythromycin;VAN,vancomycin.

第二十六页，共83页。ListofmutationsfoundinS.pneumoniaeisolatesselectedforlinezolidresistance

第二十七页，共83页。MonitoringmRNAexpressionbyqRT-PCRinlinezolidresistantStreptococcuspneumoniae第二十八页，共83页。

FunctionalanalysisofS.pneumoniaegenesinantibioticresistance

第二十九页，共83页。β-内酰胺类抗生素(β-lactamantibiotics)是指化学结构中含有β-内酰胺环的一类抗生素。分类

青霉素类头孢菌素类非典型β-内酰胺类抗菌机制抑制细菌细胞壁的合成。酶解抗生素第三十页，共83页。β-内酰胺酶β-内酰胺水解第三十一页，共83页。新型金属β-内酰胺的发现建立文库筛选阳性克隆测序接合或转化实验质粒提取测序Yongetal.2009Dec.第三十二页，共83页。IdentifytheNDM-1geneTheplasmidcarryingblaNDM-1alsocarriesblaCMY-4andthecomplexclass1integroncarryingseveralantibioticresistance-conferringgenes，andithasalsoshownitselftonaturallyhaveabroadhostrange.第三十三页，共83页。抗生素靶点修饰通过对抗生素靶点的修饰作用，抗生素的靶点变为一种不敏感的形式，从而产生了对抗生素的耐药性。大环内酯抗生素红霉素Macrolidesareproteinsynthesisinhibitors.Themechanismofactionofmacrolidesisinhibitionofbacterialproteinbiosynthesis,andtheyarethoughttodothisbypreventingpeptidyltransferasefromaddingthepeptidylattachedtotRNAtothenextaminoacidaswellasinhibitingribosomaltranslocation.第三十四页，共83页。大环内酯类抗生素的抗药机制Genotypesofmacrolideresistance:erm(erythromycinribosomalmethylase)Highlevelresistance(MIC>64)mef(specificmacrolideeffulxpump)lowlevelresistance(MIC4-8)Ermbmethylatingasingleadenineresidueofthe23SrRNABussiereetal.Biochemistry1998第三十五页，共83页。SensitivityTestof185

StreptococcuspneumoniaeIsolatesShenetal.2008分离自北京儿童的Streptococcuspneumoniae

对大环内酯抗生素的耐药情况第三十六页，共83页。erm(B)-carryingelements第三十七页，共83页。第三十八页，共83页。第三十九页，共83页。第四十页，共83页。微生物抗药机制基因水平转移环境中的抗性基因第四十一页，共83页。NewResistantBacteriaMutationsXXSusceptibleBacteriaResistantBacteriaResistanceGeneTransferEmergenceofAntimicrobialResistance第四十二页，共83页。基因水平转移是微生物进化的重要动力基因水平转移HorizontalGeneTransferThemovementofgeneticmaterialbetweentwoorganisms.Onceincorporateditisthen‘vertically’inherited.第四十三页，共83页。DelwicheandPalmer(1996)-hypothesesofHGTs1-Cyanobacteria→γ-Proteobacteria2-α-Proteobacteria→Redandbrownalgae3-γ-Proteobacteria→

α-Proteobacteria4-γ-Proteobacteria→

β-Proteobacteria第四十四页，共83页。Pan-genome泛基因组Thepan-genomeincludesthe“coregenome”containinggenespresentinallstrains,a“dispensablegenome”containinggenespresentintwoormorestrains,andfinally“uniquegenes”specifictosinglestrains。(Touchonetal.,2009).Inmolecularbiologyapan-genomedescribesthefullcomplementofgenesinaspecies(typicallyappliedtobacteriaandarchaea,whichcanhavelargevariationingenecontentamongcloselyrelatedstrains).Itisasupersetofallthegenesinallthestrainsofaspecies。第四十五页，共83页。基因水平转移的方式Transformation-nakedDNA,shortpieces,commoninbacteriathattransform.Transduction–phage,donor/recipientsharereceptors,closelyrelatedbacteria,DNA:amountinphagehead.Conjugation-plasmids/transposons,celltocellcontact,distantrelations,longDNA.第四十六页，共83页。转化TransformationCelldeath&DNAfragmentsreleasedintotheenvironmentDNAuptakebyrelatedrecipientspeciesDNAintegrationwithrecipientschromosome第四十七页，共83页。IntegrationofTransformingDNADNAiseithertakenupsingle-strandedordsDNAistakenupandonestrandisdegradedssDNA.Next,thessDNAassociateswithcompetence-specificproteinthatremainsattachedtotheDNAtoprotectitfromnucleaseattackuntilitreachesthechromosome,whereRecAtakesover.DNAisthenintegratedintothegenomeoftherecipientbyrecombination.DuringreplicationofthisheteroduplexDNA,oneparentalandonerecombinantDNAmoleculeareformed.Onsegregationatcelldivision,therecombinantDNAmoleculeispresentinthetransformedcell.第四十八页，共83页。CompetentCellsCompetentcells:abletotakeupamoleculeofDNAviatransformation.Onlycertainstrainsarecompetentandthisabilityisgeneticallydetermined.CompetenceisregulatedwithspecialproteinsplayingaroleinDNAuptakeandprocessing.ssDNAordsDNAmaytakenupbycells,thoughitmustbeinssDNAformtobeincorporatedintothegenomebyrecombination.Competentcellsbindupto1000XmoreDNAthannoncompetentcells(dsDNAbindsbettertocells).DNAfragmentscompetewitheachotherforuptake.Whilethemax.frequencyoftransformation=20%ofthepopulation,actualvalues=0.1-1.0%.Min.conc.ofDNAyieldingdetectabletransformants=0.00001g/ml.第四十九页，共83页。Transformationundernaturalconditions:Mosthuman/animalmicrobesnotnaturallycompetent

(exceptionStreptococcus&Neisseria)Soil&watermicrobesnaturallycompetent;5%ofcultivablesoilmicrobesarecompetent.”SchematicrepresentationofcompetenceregulationinStreptococcuspneumoniaeJohnsborgetal.FEMSMicrobiolRev33，2009第五十页，共83页。可以获得天然感受态造成了Spneumoniae的多样性，可利用这一特性进行研究。第五十一页，共83页。转导Transduction:Bacteriophage(phage)mediatedgenetransferBacterium(host)phagespecificinteraction:phageattachestohostcell’ssurfacereceptors&injectsDNAleavingthecapsidoutside.Insidethecell,DNAcaneither:(a)reproducetoformphageandlysethehosttoreleasethephageprogeny(processcalledlytic)OR(b)integrateintothehostgenome(processcalledlysogeny).SomephageprogenyreleasedfromthelyticmaycontainhostDNA(transducingphage)whichistransferedintoanewhostinthenextinfectioncycle.第五十二页，共83页。Generalisedtransduction第五十三页，共83页。接合Conjugation第五十四页，共83页。ConjugationCelltocellcontactbetweenadonorandarecipientcellinwhichgenesaretransferred.第五十五页，共83页。第五十六页，共83页。微生物耐药的原因耐药靶位点基因突变获得抗药基因基因水平转移（Horizontalgenetransfer）可移动遗传元件（Mobilegeneticelements）第五十七页，共83页。Integrons整合子Hall和ColhS将整合子定义为，由一系列遗传元件构成的能够识别和捕获移动性基因盒的位点特异性重组系统。一个整合子包括编码整合酶(inil)的基因及其邻近的重组识别位点(attl)。整合子不一定包括基因盒，但当基因盒整合入整合子，这些基因盒就成为整合子的一部分。绝大多数基因盒本身没有启动子序列，基因盒都按照相同的阅读框方向插入到整合子中，因此可以依赖整合子5’保守区内的启动子。第五十八页，共83页。Morethan85differentgenecassettesencodingantibioticresistancehavebeenfoundinintegrons.ThesecassettesallowtoresisttoallclassesofantibioticsusedagainsthumanGram-negativepathogens(β-lactams,aminoglycosides,chloramphenicol,trimethoprim,streptothricin,rifampin,erythromycin,…antiseptics).RecombinationReactionsinintegrons第五十九页，共83页。IntegroncassettestructureandcharacteristicsStokes,H.andHall,R(1989)MolMicro3:1669;Collis,C.andHall,R.(1992)MolMicro6:2875;Recchia,G.andHall(1995)Microbiol141:3015attCsiteICSCS(RYYYAAC)(G/TTRRRY)variableregion• allcontainanattCsite(59-baseelement)-integrasetarget• theICSisalwayscomplementarytotheCSofthecircularizedcassette;theyformimperfectinvertedrepeats• mostcontainasingleORF• promoterless第六十页，共83页。rplTrpmIinfCL20L35IF3integraseintI4orVchintIAMostare95%identicalcoresiteinversecoresiteRYYYAACGTTRRRY121-123nt:VCR126Kb,179cassettes,3%ofgenomestomrhATheV.choleraesuperintegronIntegronMazel,Detal(1998)Science280:605;Heidelberg,Jetal.(2000)Nature406:477;Rowe-Magnusetal.(1999)ResMic150:641第六十一页，共83页。微生物抗药机制基因水平转移环境中的抗性基因第六十二页，共83页。spore-formingbacteria20JANUARY2006VOL311SCIENCE自然环境是抗性基因的储存库第六十三页，共83页。Antibiotic-resistanceprofilingof75clonalisolatescapableofsubsistingonantibiotics.GautamDantas,etal.Science2008第六十四页，共83页。GlycopeptideAntibioticResistanceGenesinGlycopeptide-ProducingOrganismsMARSHALLetal.1998第六十五页，共83页。Geneticexchangeamongbacterialspecies

Thisprocessdemonstratestheimportanceofbacterialreservoirsofresistance,includingbothpathogenicandnonpathogenicorganisms.

Silbergeldetal.2008第六十六页，共83页。WW,wastewatereffluent;RWD,downstreamriverwater;RWU,upstreamriverwater.APPLIEDANDENVIRONMENTALMICROBIOLOGY,June2010,p.3444–3451抗生素污染加速了耐药性的传播和蔓延第六十七页，共83页。

养殖业是抗生素重要的污染源WorldHealthOrganizationestimateshalfoftotalamountofantimicrobialsproducedgloballyareusedinfoodanimals.InUS,70-80%ofallantimicrobialssoldareforlivestockand85%oflivestockantimicrobialuseisfornon-therapeuticfeedaddition.Diseasecontrolandgrowthpromotion第六十八页，共83页。第六十九页，共83页。WasteTreatmentandAntimicrobialsAntimicrobialsarecomplexcompoundsthatresistbiologicaldecompositionwastetreatment.Anaerobicdigestiondestroyedonly59%ofoxytetracyclineinmanuresin64days.Methaneproductionwasreducedfrom20-80%whenmanurescontainantibiotics,dependingontheconcentrationofantibioticsinthemanures.**Compostingdestroyed95%ofoxytetracylineinmanureswithinfirstweek.Antibioticsfoundintactintreatedsewagesludgewereciprofloxacin,doxycycline,norfloxacin,ofloxacin,andtriclosan.****J.Fick,et.al.,AntivialOsetimiverisnotRemovedorDegradedinNormalSewageTreatment,2007**O.A.Arikan,et.al.,FateandEffectofOxytetracyclineduringAnaerobicDigestionofManurefromTherapeuticallyTreatedCalves.,2006***E.Z.Harrison,et.al.,OrganicChemicalsinSewageSludges,2006****O.A.Arikan,et.al,CompostingRapidlyReducesLevelsofExtractableOxytetracyclineinManurefromTherapeuticallyTreatedBeefCalves,2005.第七十页，共83页。WasteTreatmentandAntimicrobialsAntimicrobialsarecomplexcompoundsthatresistbiologicaldecompositionwastetreatment.Anaerobicdigestiondestroyedonly59%ofoxytetracyclineinmanuresin64days.Methaneproductionwasreducedfrom20-80%whenmanurescontainantibiotics,dependingontheconcentrationofantibioticsinthemanures.**Compostingdestroyed95%ofoxytetracylineinmanureswithinfirstweek.Also,levelsofoxytetracyclineresistantbacteriawere10-foldlower.****Antibioticsfoundintactintreatedsewagesludgewereciprofloxacin,doxycycline,norfloxacin,ofloxacin,andtriclosan.****J.Fick,et.al.,AntivialOsetimiverisnotRemovedorDegradedinNormalSewageTreatment,2007**O.A.Arikan,et.al.,FateandEffectofOxytetracyclineduringAnaerobicDigestionofManurefromTherapeuticallyTreatedCalves.,2006***E.Z.Harrison,et.al.,OrganicChemicalsinSewageSludges,2006****O.A.Arikan,et.al,CompostingRapidlyReducesLevelsofExtractableOxytetracyclineinManurefromTherapeuticallyTreatedBeefCalves,2005.第七十一页，共83页。TetracyclineResistomeoftheOrganicPigGutKatarzynaetal.AEM,2009Of9,000bacterialartificialchromosomeclonesanalyzed,10wereidentifiedascarryingtheknowntet(C),tet(W),andtet(40)genes,aswellasnovelgenesencodingresistancetothetetracyclinesminocyclineanddoxycycline.第七十二页，共83页。TetracyclinesandTetracyclineResistanceinAgriculturalSoilsSchmittetal.2006第七十三页，共83页。象山港网箱养殖区沉积物中细菌生态分布采样地图Figure1Diversityoftheisolates第七十四页，共83页。象山港网箱养殖区沉积物中细菌抗药情况严重Figure2Antibiotics-resistanceoftheisolatesα-proteobacteriaγ-proteobacteriaActinobacteriaFirmicutesBacteroidetes第七十五页，共83页。分离到的耐药细菌所含质粒多样12345678910111213141D15000DNALadder2,9Comamonassp.CNB-13,8GemoneDNA4Pseudoalteromonashaloplanktis

5Exiguobacteriumsp.

6Pseudoclavibacterhelvolus

7Exiguobacteriumaestuarii

10Roseobactergallaeciensis11Roseobactergallaeciensis12Brevundimonasnasdae13Phaeobactercaeruleus14Labrenziaaggregata

第七十六页，共83页。Planococcuscitreus

Bacteria;Firmicutes;Bacillales;Planococcaceae;PlanococcusPlanococcuscitreusbacillussubtilisMIC提高了8倍EnterococcusFaecom结合转移第七十七页，共83页。plasmidsisolatedfromtheabovestrainswereusedfornaturaltransformationoftheo