微生物抗藥性及生態(tài)效應(yīng)

微生物抗藥性及生態(tài)效應(yīng)第一頁，共83頁。微生物抗藥機(jī)制基因水平轉(zhuǎn)移環(huán)境中的抗性基因第二頁，共83頁。MycobacteriumtuberculosisEnterococcusfaecalisStreptococcuspneumoniaeE.coli第三頁，共83頁。第四頁，共83頁?？股乜顾幮砸呀?jīng)成為全球性的重大問題

Resistance1998-2004-%%Resistance1998-2004-%StreptococcuspneumoniaeinUSA第五頁，共83頁。第六頁，共83頁。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第七頁，共83頁。EscherichiacoliinAustralia201011KlebsiellapneumoniaeIndia200912Kpneumoniae，EcoliIndia,Pakistan,andtheUK20108CitrobacterfreundiiFrench

returningfromIndia201010屎腸球菌，鮑曼不動(dòng)桿菌201010超級(jí)細(xì)菌（Superbug）NDM-1NewMetallo--LactamaseNDM-1超級(jí)細(xì)菌在世界各地被發(fā)現(xiàn)。第八頁，共83頁。我國發(fā)現(xiàn)NDM-1超級(jí)細(xì)菌第九頁，共83頁。第十頁，共83頁。超級(jí)細(xì)菌對(duì)多種抗生素均有抗性第十一頁，共83頁。Tigecycline

新批準(zhǔn)的新型抗生素

[簡介]：Tigecycline(商品名為Tygacil；惠氏公司生產(chǎn))2005年6月獲得FDA批準(zhǔn)，用于治療細(xì)菌感染。這是一種稱為glycylcyclines的新型抗生素第一個(gè)獲得批準(zhǔn)的產(chǎn)品。該類抗生素用于克服早期四環(huán)素類抗生素的耐藥性。除了Tigecycline，在過去的40年里，只有兩個(gè)新型抗生素上市，分別是輝瑞公司的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，主要針對(duì)革蘭氏陰性桿菌。第十二頁，共83頁。第十三頁，共83頁。超級(jí)細(xì)菌（Superbug）Plasmids

Chennai50kbto350kb；Haryana118kb(54%)or50kb(36%)；UKisolates80kbtogreaterthan500kb.MostblaNDM-1positiveplasmidswerereadilytransferableandpronetorearrangement,losingor(morerarely)gainingDNAontransfer.質(zhì)粒的可移動(dòng)性和可塑性可能意味著在細(xì)菌中的廣泛傳播和不斷變異。第十四頁，共83頁。加拿大魁北克省舍布魯克大學(xué)附屬醫(yī)院的專家日前透露，從2003年年初至今，一種普通的腸道細(xì)菌——梭狀芽孢桿菌歷經(jīng)兩年變異，已成為致命的“超級(jí)病菌”，它可引起65歲以上老年人和服用抗生素的病人產(chǎn)生嚴(yán)重痢疾，并最終致死。該病菌已使這家醫(yī)院的100名病人死亡，如不采取緊急行動(dòng)，這種“超級(jí)病菌”很可能會(huì)引發(fā)一場(chǎng)致命傳染病的蔓延。

第十五頁，共83頁。2010年9月5日，日本媒體近日披露，有46名患者在日本帝京大學(xué)醫(yī)學(xué)部附屬醫(yī)院住院期間感染了幾乎可以抵抗所有抗生素的超級(jí)細(xì)菌——多重耐藥鮑曼不動(dòng)桿菌，其中9名患者的死亡因感染此種病菌所致。該醫(yī)院曾刻意隱瞞感染事件，導(dǎo)致日本首次出現(xiàn)大規(guī)模不動(dòng)桿菌感染，日本警方遂決定傳喚該院醫(yī)生，展開調(diào)查。經(jīng)檢測(cè)發(fā)現(xiàn)，患者感染的不動(dòng)桿菌能抵抗多種抗生素，目前只有先鋒霉素族的一部分抗生素對(duì)這一病菌有效。

第十六頁，共83頁。-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第十七頁，共83頁。細(xì)菌耐藥機(jī)制DrugImpermeabilityDrugEffluxTargetmodificationDrugInactivationOnetypeofantibioticcanbeovercomebydifferentmechanismsDifferentclassesofantibioticscanhavethesamemechanismsofresistance第十八頁，共83頁。孔蛋白改變，細(xì)胞壁/膜通透性改變?nèi)f古霉素（vancomycin）：由一種鏈霉菌產(chǎn)生的、結(jié)構(gòu)復(fù)雜的糖肽類抗生素，專一地抑制肽聚糖的生物合成。萬古霉素通過抑制細(xì)菌的生長和繁殖來殺死細(xì)菌。這種藥物通過干擾細(xì)菌細(xì)胞壁結(jié)構(gòu)中的一種關(guān)鍵組分來干擾細(xì)胞壁的合成，抑制細(xì)胞壁中磷脂和多肽的生成，只是針對(duì)革蘭氏陽性菌，對(duì)厭氧菌和革蘭氏陰性細(xì)菌無效。萬古霉素的藥力較強(qiáng)，且有腎毒性，在其他抗生素對(duì)病菌無效時(shí)才會(huì)被使用，也就是所謂的最后一線藥物。

第十九頁，共83頁。Howdenetal.2006VISAstrainshavethickercellwalls第二十頁，共83頁。-adsorptionofVnbycell-wallD-ala-D-alaresiduesincreased,diffusiondecreased-drugispreventedfromreachingitsactivesiteVnsensitiveVnresistantVISAstrainshavethickercellwalls第二十一頁，共83頁。TheCaseofCMRSA-1MRSAVISA(4mg/L)potentialhVISA(<4mg/L)第二十二頁，共83頁。TheCaseofCMRSA-1敲除株野生型耐藥株45.7+/-6.722.1+/-2.529.0+/-3.2JolyneDrummelsmith,JournalofProteomeResearch2007,6,4690–4702第二十三頁，共83頁。主動(dòng)外排泵因?yàn)橥馀疟玫闹鲃?dòng)輸出抗生素，因而細(xì)菌內(nèi)達(dá)不到治療濃度。一些泵具有相對(duì)較強(qiáng)的特異性，如四環(huán)素運(yùn)輸泵，而其他輸出泵具有廣泛的底物選擇性和耐藥性。細(xì)菌體內(nèi)具有大量的輸出泵，用于運(yùn)輸代謝物和外來有毒物質(zhì)。第二十四頁，共83頁。Linezolid-oxazolidinone第二十五頁，共83頁。SusceptibilitylevelsofS.pneumoniaeisolates

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

第二十六頁，共83頁。ListofmutationsfoundinS.pneumoniaeisolatesselectedforlinezolidresistance

第二十七頁，共83頁。MonitoringmRNAexpressionbyqRT-PCRinlinezolidresistantStreptococcuspneumoniae第二十八頁，共83頁。

FunctionalanalysisofS.pneumoniaegenesinantibioticresistance

第二十九頁，共83頁。β-內(nèi)酰胺類抗生素(β-lactamantibiotics)是指化學(xué)結(jié)構(gòu)中含有β-內(nèi)酰胺環(huán)的一類抗生素。分類

青霉素類頭孢菌素類非典型β-內(nèi)酰胺類抗菌機(jī)制抑制細(xì)菌細(xì)胞壁的合成。酶解抗生素第三十頁，共83頁。β-內(nèi)酰胺酶β-內(nèi)酰胺水解第三十一頁，共83頁。新型金屬β-內(nèi)酰胺的發(fā)現(xiàn)建立文庫篩選陽性克隆測(cè)序接合或轉(zhuǎn)化實(shí)驗(yàn)質(zhì)粒提取測(cè)序Yongetal.2009Dec.第三十二頁，共83頁。IdentifytheNDM-1geneTheplasmidcarryingblaNDM-1alsocarriesblaCMY-4andthecomplexclass1integroncarryingseveralantibioticresistance-conferringgenes，andithasalsoshownitselftonaturallyhaveabroadhostrange.第三十三頁，共83頁?？股匕悬c(diǎn)修飾通過對(duì)抗生素靶點(diǎn)的修飾作用，抗生素的靶點(diǎn)變?yōu)橐环N不敏感的形式，從而產(chǎn)生了對(duì)抗生素的耐藥性。大環(huán)內(nèi)酯抗生素紅霉素Macrolidesareproteinsynthesisinhibitors.Themechanismofactionofmacrolidesisinhibitionofbacterialproteinbiosynthesis,andtheyarethoughttodothisbypreventingpeptidyltransferasefromaddingthepeptidylattachedtotRNAtothenextaminoacidaswellasinhibitingribosomaltranslocation.第三十四頁，共83頁。大環(huán)內(nèi)酯類抗生素的抗藥機(jī)制Genotypesofmacrolideresistance:erm(erythromycinribosomalmethylase)Highlevelresistance(MIC>64)mef(specificmacrolideeffulxpump)lowlevelresistance(MIC4-8)Ermbmethylatingasingleadenineresidueofthe23SrRNABussiereetal.Biochemistry1998第三十五頁，共83頁。SensitivityTestof185

StreptococcuspneumoniaeIsolatesShenetal.2008分離自北京兒童的Streptococcuspneumoniae

對(duì)大環(huán)內(nèi)酯抗生素的耐藥情況第三十六頁，共83頁。erm(B)-carryingelements第三十七頁，共83頁。第三十八頁，共83頁。第三十九頁，共83頁。第四十頁，共83頁。微生物抗藥機(jī)制基因水平轉(zhuǎn)移環(huán)境中的抗性基因第四十一頁，共83頁。NewResistantBacteriaMutationsXXSusceptibleBacteriaResistantBacteriaResistanceGeneTransferEmergenceofAntimicrobialResistance第四十二頁，共83頁。基因水平轉(zhuǎn)移是微生物進(jìn)化的重要?jiǎng)恿蛩睫D(zhuǎn)移HorizontalGeneTransferThemovementofgeneticmaterialbetweentwoorganisms.Onceincorporateditisthen‘vertically’inherited.第四十三頁，共83頁。DelwicheandPalmer(1996)-hypothesesofHGTs1-Cyanobacteria→γ-Proteobacteria2-α-Proteobacteria→Redandbrownalgae3-γ-Proteobacteria→

α-Proteobacteria4-γ-Proteobacteria→

β-Proteobacteria第四十四頁，共83頁。Pan-genome泛基因組Thepan-genomeincludesthe“coregenome”containinggenespresentinallstrains,a“dispensablegenome”containinggenespresentintwoormorestrains,andfinally“uniquegenes”specifictosinglestrains。(Touchonetal.,2009).Inmolecularbiologyapan-genomedescribesthefullcomplementofgenesinaspecies(typicallyappliedtobacteriaandarchaea,whichcanhavelargevariationingenecontentamongcloselyrelatedstrains).Itisasupersetofallthegenesinallthestrainsofaspecies。第四十五頁，共83頁?；蛩睫D(zhuǎn)移的方式Transformation-nakedDNA,shortpieces,commoninbacteriathattransform.Transduction–phage,donor/recipientsharereceptors,closelyrelatedbacteria,DNA:amountinphagehead.Conjugation-plasmids/transposons,celltocellcontact,distantrelations,longDNA.第四十六頁，共83頁。轉(zhuǎn)化TransformationCelldeath&DNAfragmentsreleasedintotheenvironmentDNAuptakebyrelatedrecipientspeciesDNAintegrationwithrecipientschromosome第四十七頁，共83頁。IntegrationofTransformingDNADNAiseithertakenupsingle-strandedordsDNAistakenupandonestrandisdegradedssDNA.Next,thessDNAassociateswithcompetence-specificproteinthatremainsattachedtotheDNAtoprotectitfromnucleaseattackuntilitreachesthechromosome,whereRecAtakesover.DNAisthenintegratedintothegenomeoftherecipientbyrecombination.DuringreplicationofthisheteroduplexDNA,oneparentalandonerecombinantDNAmoleculeareformed.Onsegregationatcelldivision,therecombinantDNAmoleculeispresentinthetransformedcell.第四十八頁，共83頁。CompetentCellsCompetentcells:abletotakeupamoleculeofDNAviatransformation.Onlycertainstrainsarecompetentandthisabilityisgeneticallydetermined.CompetenceisregulatedwithspecialproteinsplayingaroleinDNAuptakeandprocessing.ssDNAordsDNAmaytakenupbycells,thoughitmustbeinssDNAformtobeincorporatedintothegenomebyrecombination.Competentcellsbindupto1000XmoreDNAthannoncompetentcells(dsDNAbindsbettertocells).DNAfragmentscompetewitheachotherforuptake.Whilethemax.frequencyoftransformation=20%ofthepopulation,actualvalues=0.1-1.0%.Min.conc.ofDNAyieldingdetectabletransformants=0.00001g/ml.第四十九頁，共83頁。Transformationundernaturalconditions:Mosthuman/animalmicrobesnotnaturallycompetent

(exceptionStreptococcus&Neisseria)Soil&watermicrobesnaturallycompetent;5%ofcultivablesoilmicrobesarecompetent.”SchematicrepresentationofcompetenceregulationinStreptococcuspneumoniaeJohnsborgetal.FEMSMicrobiolRev33，2009第五十頁，共83頁?？梢垣@得天然感受態(tài)造成了Spneumoniae的多樣性，可利用這一特性進(jìn)行研究。第五十一頁，共83頁。轉(zhuǎn)導(dǎo)Transduction:Bacteriophage(phage)mediatedgenetransferBacterium(host)phagespecificinteraction:phageattachestohostcell’ssurfacereceptors&injectsDNAleavingthecapsidoutside.Insidethecell,DNAcaneither:(a)reproducetoformphageandlysethehosttoreleasethephageprogeny(processcalledlytic)OR(b)integrateintothehostgenome(processcalledlysogeny).SomephageprogenyreleasedfromthelyticmaycontainhostDNA(transducingphage)whichistransferedintoanewhostinthenextinfectioncycle.第五十二頁，共83頁。Generalisedtransduction第五十三頁，共83頁。接合Conjugation第五十四頁，共83頁。ConjugationCelltocellcontactbetweenadonorandarecipientcellinwhichgenesaretransferred.第五十五頁，共83頁。第五十六頁，共83頁。微生物耐藥的原因耐藥靶位點(diǎn)基因突變獲得抗藥基因基因水平轉(zhuǎn)移（Horizontalgenetransfer）可移動(dòng)遺傳元件（Mobilegeneticelements）第五十七頁，共83頁。Integrons整合子Hall和ColhS將整合子定義為，由一系列遺傳元件構(gòu)成的能夠識(shí)別和捕獲移動(dòng)性基因盒的位點(diǎn)特異性重組系統(tǒng)。一個(gè)整合子包括編碼整合酶(inil)的基因及其鄰近的重組識(shí)別位點(diǎn)(attl)。整合子不一定包括基因盒，但當(dāng)基因盒整合入整合子，這些基因盒就成為整合子的一部分。絕大多數(shù)基因盒本身沒有啟動(dòng)子序列，基因盒都按照相同的閱讀框方向插入到整合子中，因此可以依賴整合子5’保守區(qū)內(nèi)的啟動(dòng)子。第五十八頁，共83頁。Morethan85differentgenecassettesencodingantibioticresistancehavebeenfoundinintegrons.ThesecassettesallowtoresisttoallclassesofantibioticsusedagainsthumanGram-negativepathogens(β-lactams,aminoglycosides,chloramphenicol,trimethoprim,streptothricin,rifampin,erythromycin,…antiseptics).RecombinationReactionsinintegrons第五十九頁，共83頁。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第六十頁，共83頁。rplTrpmIinfCL20L35IF3integraseintI4orVchintIAMostare95%identicalcoresiteinversecoresiteRYYYAACGTTRRRY121-123nt:VCR126Kb,179cassettes,3%ofgenomestomrhATheV.choleraesuperintegronIntegronMazel,Detal(1998)Science280:605;Heidelberg,Jetal.(2000)Nature406:477;Rowe-Magnusetal.(1999)ResMic150:641第六十一頁，共83頁。微生物抗藥機(jī)制基因水平轉(zhuǎn)移環(huán)境中的抗性基因第六十二頁，共83頁。spore-formingbacteria20JANUARY2006VOL311SCIENCE自然環(huán)境是抗性基因的儲(chǔ)存庫第六十三頁，共83頁。Antibiotic-resistanceprofilingof75clonalisolatescapableofsubsistingonantibiotics.GautamDantas,etal.Science2008第六十四頁，共83頁。GlycopeptideAntibioticResistanceGenesinGlycopeptide-ProducingOrganismsMARSHALLetal.1998第六十五頁，共83頁。Geneticexchangeamongbacterialspecies

Thisprocessdemonstratestheimportanceofbacterialreservoirsofresistance,includingbothpathogenicandnonpathogenicorganisms.

Silbergeldetal.2008第六十六頁，共83頁。WW,wastewatereffluent;RWD,downstreamriverwater;RWU,upstreamriverwater.APPLIEDANDENVIRONMENTALMICROBIOLOGY,June2010,p.3444–3451抗生素污染加速了耐藥性的傳播和蔓延第六十七頁，共83頁。

養(yǎng)殖業(yè)是抗生素重要的污染源WorldHealthOrganizationestimateshalfoftotalamountofantimicrobialsproducedgloballyareusedinfoodanimals.InUS,70-80%ofallantimicrobialssoldareforlivestockand85%oflivestockantimicrobialuseisfornon-therapeuticfeedaddition.Diseasecontrolandgrowthpromotion第六十八頁，共83頁。第六十九頁，共83頁。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.第七十頁，共83頁。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.第七十一頁，共83頁。TetracyclineResistomeoftheOrganicPigGutKatarzynaetal.AEM,2009Of9,000bacterialartificialchromosomeclonesanalyzed,10wereidentifiedascarryingtheknowntet(C),tet(W),andtet(40)genes,aswellasnovelgenesencodingresistancetothetetracyclinesminocyclineanddoxycycline.第七十二頁，共83頁。TetracyclinesandTetracyclineResistanceinAgriculturalSoilsSchmittetal.2006第七十三頁，共83頁。象山港網(wǎng)箱養(yǎng)殖區(qū)沉積物中細(xì)菌生態(tài)分布采樣地圖Figure1Diversityoftheisolates第七十四頁，共83頁。象山港網(wǎng)箱養(yǎng)殖區(qū)沉積物中細(xì)菌抗藥情況嚴(yán)重Figure2Antibiotics-resistanceoftheisolatesα-proteobacteriaγ-proteobacteriaActinobacteriaFirmicutesBacteroidetes第七十五頁，共83頁。分離到的耐藥細(xì)菌所含質(zhì)粒多樣12345678910111213141D15000DNALadder2,9Comamonassp.CNB-13,8GemoneDNA4Pseudoalteromonashaloplanktis

5Exiguobacteriumsp.

6Pseudoclavibacterhelvolus

7Exiguobacteriumaestuarii

10Roseobactergallaeciensis11Roseobactergallaeciensis12Brevundimonasnasdae13Phaeobactercaeruleus14Labrenziaaggregata

第七十六頁，共83頁。Planococcuscitreus

Bacteria;Firmicutes;Bacillales;Planococcaceae;PlanococcusPlanococcuscitreusbacillussubtilisMIC提高了8倍EnterococcusFaecom結(jié)合轉(zhuǎn)移第七十七頁，共83頁。plasmidsisolatedfromtheabovestrainswereusedfornaturaltransformationoftheo