微生物抗药性及生态效应

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,2020/11/3,*,微生物抗药性及生态效应,冯 婕,中国科学院微生物研究所,2020/11/3,1,微生物抗药机制,基因水平转移,环境中的抗性基因,2020/11/3,2,Mycobacterium tuberculosis,Enterococcus faecalis,Streptococcus pneumoniae,E. coli,2020/11/3,3,2020/11/3,4,抗生素抗药性已经成为全球性的重大问题,Resistance 1998-2004 - %,%,Resistance 1998-2004 - %,Streptococcus pneumoniae,in USA,2020/11/3,5,2020/11/3,6,S.,aureus,Penicillin,1950s,Penicillin-resistant,S. aureus,Evolution of Drug Resistance in,S. aureus,Methicillin,1970s,Methicillin-resistant,S. aureus,(MRSA),Vancomycin-resistant,enterococci (VRE),Vancomycin,1990s,1997,Vancomycin,intermediate-,resistant,S. aureus,(VISA), 2002 ,Vancomycin-,resistant,S. aureus,2020/11/3,7,Escherichia coli,in Australia 2010 11,Klebsiella pneumoniae,India,2009 12,K,pneumoniae,，,E coli,India, Pakistan, and the UK 2010 8,Citrobacter freundii,French,returning from India 2010 10,屎肠球菌，鲍曼不动杆菌,2010 10,超级细菌（,Super bug,）,NDM-1,New Metallo-Lactamase,NDM-1,超级细菌在世界各地被发现。,2020/11/3,8,我国发现NDM-1超级细菌,2020/11/3,9,2020/11/3,10,超级细菌对多种抗生素均有抗性,2020/11/3,11,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,(polymyxin E),是是混合物多聚氨基酸， 主要针对革兰氏阴性杆菌 。,2020/11/3,12,2020/11/3,13,超级细菌（,Super bug,）,Plasmids,Chennai 50 kb to 350 kb,；,Haryana 118 kb (54%) or 50 kb (36%),；,UK isolates 80 kb to greater than 500 kb.,Most,bla,NDM-1 positive plasmids were,readily transferable and prone to rearrangement, losing or (more rarely) gaining DNA on transfer.,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。,2020/11/3,14,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。,2020/11/3,15,2010,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。,2020/11/3,16,-MRSA infection of hip screw,-Vn MIC 1 mg/L, Vn treatment,-d55, developed,Enterobacter,infection,-cipro added to Vn for 6 weeks,-d99, Vn MIC = 4 mg/L,-d159, Vn discontinued,-d174, readmitted to hospital, Vn treatment,-Vn MIC = 4 mg/L,-d188, linezolid + rifampin,-d198, infection cleared,-12 surgeries including removal of infected bone,A case of resistant,S aureus,in patient,Canada-Edmonton-Webster et al., 2007,2020/11/3,17,细菌耐药机制,Drug Impermeability,Drug Efflux,Target modification,Drug Inactivation,One type of antibiotic can be overcome by different mechanisms,Different classes of antibiotics can have the same mechanisms of resistance,2020/11/3,18,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。,2020/11/3,19,Howden et al. 2006,VISA strains have thicker cell walls,2020/11/3,20,-adsorption of Vn by cell-wall D-ala-D-ala residues increased, diffusion decreased,-drug is prevented from reaching its active site,Vn sensitive,Vn resistant,VISA strains have thicker cell walls,2020/11/3,21,The Case of CMRSA-1,MRSA,VISA,(,4 mg/L),potential hVISA,(64),mef,(specific macrolide effulx pump ),low level resistance (MIC 4-8),Ermb methylating a single adenine,residue of the 23S rRNA,Bussiere et al.,Biochemistry,1998,2020/11/3,35,Sensitivity Test of 185,Streptococcus pneumoniae,Isolates,Shen et al. 2008,分离自北京儿童的,Streptococcus pneumoniae,对大环内酯抗生素的耐药情况,2020/11/3,36,erm,(B)-carrying elements,2020/11/3,37,2020/11/3,38,2020/11/3,39,2020/11/3,40,微生物抗药机制,基因水平转移,环境中的抗性基因,2020/11/3,41,New Resistant Bacteria,Mutations,XX,Susceptible Bacteria,Resistant Bacteria,Resistance Gene Transfer,Emergence of Antimicrobial Resistance,2020/11/3,42,基因水平转移是微生物进化的重要动力,基因水平转移,Horizontal Gene Transfer,The movement of genetic material between two organisms. Once incorporated it is then vertically inherited.,2020/11/3,43,Delwiche and Palmer (1996) - hypotheses of HGTs,1- Cyanobacteria,-,Proteobacteria,2-,-Proteobacteria, Red and brown algae,3-,-Proteobacteria ,-Proteobacteria,4-,-Proteobacteria ,-Proteobacteria,2020/11/3,44,Pan-genome,泛基因组,The pan-genome includes the “core genome” containing genes present in all strains, a “dispensable genome” containing genes present in two or more strains, and finally “unique genes” specific to single strains,。,(Touchon et al., 2009).,In molecular biology a,pan-genome,describes the full complement of,genes,in a,species,(typically applied to,bacteria,and,archaea, which can have large variation in gene content among closely related strains). It is a,superset,of all the genes in all the,strains,of a species,。,2020/11/3,45,基因水平转移的方式,Transformation,- naked DNA, short pieces, common in bacteria that transform .,Transduction, phage, donor/recipient share receptors, closely related bacteria, DNA: amount in phage head.,Conjugation,-plasmids/transposons, cell to cell contact, distant relations, long DNA.,2020/11/3,46,转化,Transformation,Cell death & DNA fragments released into the environment,DNA uptake by related recipient species,DNA integration with recipients chromosome,2020/11/3,47,Integration of Transforming DNA,DNA is either taken up single-stranded or dsDNA is taken up and one strand is degraded, ssDNA.,Next, the ssDNA associates with competence-specific protein that remains attached to the DNA to protect it from nuclease attack until it reaches the chromosome, where RecA takes over.,DNA is then integrated into the genome of the recipient by recombination.,During replication of this heteroduplex DNA, one parental and one recombinant DNA molecule are formed. On segregation at cell division, the recombinant DNA molecule is present in the transformed cell.,2020/11/3,48,Competent Cells,Competent cells,: able to take up a molecule of DNA via transformation.,Only certain strains are competent and this ability is genetically determined.,Competence is regulated with special proteins playing a role in DNA uptake and processing.,ssDNA or dsDNA may taken up by cells, though it must be in ssDNA form to be incorporated into the genome by recombination.,Competent cells bind up to 1000X more DNA than noncompetent cells (dsDNA binds better to cells).,DNA fragments compete with each other for uptake.,While the max. frequency of transformation = 20% of the population, actual values = 0.1-1.0%.,Min. conc. of DNA yielding detectable transformants = 0.00001,g/ml.,2020/11/3,49,Transformation under natural conditions,:,Most human / animal microbes not naturally,competent,(exception,Streptococcus,&,Neisseria,),Soil & water microbes naturally,competent,; 5% of cultivable soil microbes are,competent,.”,Schematic representation of competence regulation in,Streptococcus pneumoniae,Johnsborg et al. FEMS Microbiol Rev 33,，,2009,2020/11/3,50,可以获得天然感受态造成了,S pneumoniae,的多样性，可利用这一特性进行研究,。,2020/11/3,51,转导,Transduction,: Bacteriophage (phage) mediated gene transfer,Bacterium (host) phage specific interaction: phage attaches to host cells surface receptors & injects DNA leaving the capsid outside.,Inside the cell, DNA can either:,(a) reproduce to form phage and lyse the host to release the phage progeny (process called lytic) OR,(b) integrate into the host genome (process called lysogeny).,Some phage progeny released from the lytic may contain host DNA (transducing phage) which is transfered into a new host in the next infection cycle.,2020/11/3,52,Generalised transduction,2020/11/3,53,接合,Conjugation,2020/11/3,54,Conjugation,Cell to cell contact between a donor and a recipient cell in which genes are transferred.,2020/11/3,55,2020/11/3,56,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,（,Horizontal gene transfer,）,可移动遗传元件,（,Mobile genetic elements,）,2020/11/3,57,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。,2020/11/3,58,More than,85,different gene cassettes encoding antibiotic resistance have been found in integrons.,These cassettes allow to resist to,all classes of antibiotics,used against human Gram-negative pathogens,(,-lactams, aminoglycosides, chloramphenicol, trimethoprim, streptothricin, rifampin, erythromycin, antiseptics).,Recombination,Reactions in integrons,2020/11/3,59,Integron cassette structure and characteristics,Stokes, H. and Hall, R (1989) Mol Micro 3:1669; Collis, C. and Hall, R.(1992) Mol Micro 6:2875; Recchia, G. and Hall (1995) Microbiol 141:3015,attC site,ICS,CS,(RYYYAAC),(G / TTRRRY),variable region,all contain an attC site (59-base element) - integrase target,the ICS is always complementary to the CS of the circularized cassette; they form imperfect inverted repeats,most contain a single ORF,promoterless,2020/11/3,60,r,p,l,T,r,p,m,I,i,n,f,C,L,2,0,L,3,5,I,F,3,i,n,t,e,g,r,a,s,e,intI4 or,VchintIA,Most are 95% identical,core site,inverse core site,RYYYAAC,GTTRRRY,121-123 nt,: VCR,126 Kb, 179 cassettes, 3% of genome,sto,mrhA,The V. cholerae superintegron,Integron,Mazel, D et al (1998) Science 280:605; Heidelberg, Jet al. (2000) Nature 406:477; Rowe-Magnus et al. (1999) Res Mic 150:641,2020/11/3,61,微生物抗药机制,基因水平转移,环境中的抗性基因,2020/11/3,62,spore-forming bacteria,20 JANUARY 2006 VOL 311 SCIENCE,自然环境是抗性基因的储存库,2020/11/3,63,Antibiotic-resistance profiling of 75 clonal isolates capable of subsisting,on antibiotics.,Gautam Dantas,et al. Science 2008,2020/11/3,64,Glycopeptide Antibiotic Resistance Genes in Glycopeptide-Producing Organisms,MARSHALL et al. 1998,2020/11/3,65,Genetic exchange among bacterial species,This process demonstrates the importance of bacterial reservoirs of resistance, including both pathogenic and nonpathogenic organisms.,Silbergeld et al. 2008,2020/11/3,66,WW, wastewater effluent; RWD, downstream river water; RWU, upstream river water.,APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2010, p. 34443451,抗生素污染加速了耐药性的传播和蔓延,2020/11/3,67,养殖业是抗生素重要的污染源,World Health Organization estimates half of total amount of antimicrobials produced globally are used in food animals.,In US, 70-80% of all antimicrobials sold are for livestock and 85% of livestock antimicrobial use is for non-therapeutic feed addition.,Disease control and growth promotion,2020/11/3,68,2020/11/3,69,Waste Treatment and Antimicrobials,Antimicrobials are complex compounds that resist biological decomposition waste treatment.,Anaerobic digestion destroyed only 59% of oxytetracycline in manures in 64 days. Methane production was reduced from 20-80% when manures contain antibiotics, depending on the concentration of antibiotics in the manures. *,Composting destroyed 95% of oxytetracyline in manures within first week.,Antibiotics found intact in treated sewage sludge were ciprofloxacin, doxycycline, norfloxacin, ofloxacin, and triclosan.*,*J.Fick, et.al., Antivial Osetimiver is not Removed or Degraded in Normal Sewage Treatment, 2007,*O.A. Arikan, et.al., Fate and Effect of Oxytetracycline during Anaerobic Digestion of Manure from Therapeutically Treated Calves., 2006,*E.Z.Harrison, et.al., Organic Chemicals in Sewage Sludges, 2006,*O.A. Arikan, et.al, Composting Rapidly Reduces Levels of Extractable Oxytetracycline in Manure from Therapeutically Treated Beef Calves, 2005.,2020/11/3,70,Waste Treatment and Antimicrobials,Antimicrobials are complex compounds that resist biological decomposition waste treatment.,Anaerobic digestion destroyed only 59% of oxytetracycline in manures in 64 days. Methane production was reduced from 20-80% when manures contain antibiotics, depending on the concentration of antibiotics in the manures. *,Composting destroyed 95% of oxytetracyline in manures within first week. Also, levels of oxytetracycline resistant bacteria were 10-fold lower. *,Antibiotics found intact in treated sewage sludge were ciprofloxacin, doxycycline, norfloxacin, ofloxacin, and triclosan.*,*J.Fick, et.al., Antivial Osetimiver is not Removed or Degraded in Normal Sewage Treatment, 2007,*O.A. Arikan, et.al., Fate and Effect of Oxytetracycline during Anaerobic Digestion of Manure from Therapeutically Treated Calves., 2006,*E.Z.Harrison, et.al., Organic Chemicals in Sewage Sludges, 2006,*O.A. Arikan, et.al, Composting Rapidly Reduces Levels of Extractable Oxytetracycline in Manure from Therapeutically Treated Beef Calves, 2005.,2020/11/3,71,Tetracycline Resistome of the Organic Pig Gut,Katarzyna et al. AEM, 2009,Of 9,000 bacterial artificial chromosome clones analyzed, 10 were identified as carrying the known,tet,(C),tet,(W), and,tet,(40) genes, as well as novel genes encoding resistance to the,tetracyclines minocycline and doxycycline.,2020/11/3,72,Tetracyclines and Tetracycline,Resistance in Agricultural Soils,Schmitt et al. 2006,2020/11/3,73,象山港网箱养殖区沉积物中细菌生态分布,采样地图,Figure 1 Diversity of the isolates,2020/11/3,74,象山港网箱养殖区沉积物中细菌抗药情况严重,Figure 2 Antibiotics-resistance of the isolates,-proteobacteria - proteobacteria Actinobacteria Firmicutes Bacteroidetes,2020/11/3,75,分离到的耐药细菌所含质粒多样,1 2 3 4 5 6 7 8 9 10 11 12 13 14,1 D15000 DNA Ladder,2,9,Comamonas sp.,CNB-1,3,8 Gemone DNA,4,Pseudoalteromonas haloplanktis,5,Exiguobacterium sp.,6,Pseudoclavibacter helvolus,7,Exiguobacterium aestuarii,10,Roseobacter gallaeciensis,11,Roseobacter gallaeciensis,12,Brevundimonas nasdae,13,Phaeobacter caeruleus,14,Labrenzia aggregata,2020/11/3,76,Planococ,cus citreus,Bacteria; Firmicutes; Bacillales; Planococcaceae; Planococcus,Planococ,cus citreus,bacillus subtilis,MIC,提高了,8,倍,Enterococcus Faecom,结合转移,2020/11/3,77,plasmids isolated from the above strains were used for natural transformation of the oral cariogenic pathogen,Streptococcus mutans,under laboratory conditions. The tetS/M gene was successfully transferred to S. mutans UA159 at frequencies ranging from 1.910,-7,to 2.810,-5, 4.710,-7,to 2.310,-6, and 3.810,-7,to 2.110,-6,transformants per recipient cell using CZ-T4, CZ-T8 and RMKT14 plasmid extracts, respectively.,食品中也存在抗性基因，,并可通过基因水平转移传递给口腔致病菌,FEMS Microbiol Lett 254 (2006) 226231,2020/11/3,78,Most of the resistance genes we identified using culture-independent sampling have not been previously identified and are evolutionarily distant from known resistance genes.,Nearly half of the resistance genes we identified in cultured aerobic gut isolates (a small subset of the gut microbiome) are identical to resistance genes harbored by major pathogens.,The immense diversity of resistance genes in the human microbiome could contribute to future emergence of antibiotic resistance in human pathogens.,Antibiotic resistance profiles of cultured aerobic gut microbiome isolates,Sommer et al. Science 2009,Antibiotic,Resistance Reservoir in the Human Microflora,2020/11/3,79,Map of NDM-1-positive samples from New Delhi centre and surrounding areas,2020/11/3,80,Minimal inhibitory concentration of antimicrobials and genetic characteristics of NDM-1-positive bacteria,Timothy,et al,. 2011,2020/11/3,81,Transfer of antibiotic resistance genes from natural clinical environments.,Most antibiotic resistance genes acquired through horizontal gene transfer have been originated in environmental microorganisms. They suffer as the consequence of human activities,might be relevant for the future evolution and dissemination of antibiotic resistance determinants in bacterial pathogens.,研究环境中的抗性基因具有非常重要的意义,2020/11/3,82,谢谢,2020/11/3,83,