1、微生物抗藥性及生態效應微生物抗藥機制微生物抗藥機制基因水平轉移基因水平轉移環境中的抗性基因環境中的抗性基因Mycobacterium tuberculosisEnterococcus faecalisStreptococcus pneumoniaeE. coli抗生素抗藥性已經成為全球性的重大問題抗生素抗藥性已經成為全球性的重大問題 Resistance 1998-2004 - %Resistance 1998-2004 - %Streptococcus pneumoniae in USAS. aureusPenicillin1950sPenicillin-resistantS. aureus

2、Methicillin1970sMethicillin-resistant S. aureus (MRSA)Vancomycin-resistantenterococci (VRE)Vancomycin1990s1997Vancomycinintermediate-resistantS. aureus (VISA) 2002 Vancomycin-resistantS. aureusEscherichia coli in Australia 2010 11Klebsiella pneumoniae India2009 12K pneumoniae， E coli India, Pakistan

3、, and the UK 2010 8Citrobacter freundii French returning from India 2010 10屎腸球菌，鮑曼不動桿菌屎腸球菌，鮑曼不動桿菌 2010 10超級細菌（超級細菌（Super bug）NDM-1New Metallo-LactamaseNDM-1超級細菌在世界各地被發現。超級細菌在世界各地被發現。超級細菌對多種抗生素均有抗性超級細菌對多種抗生素均有抗性Tigecycline 新批準的新型抗生素 簡介：Tigecycline(商品名為Tygacil；惠氏公司生產)2005年6月獲得FDA批準，用于治療細菌感染。這是一種稱為glyc

4、ylcyclines的新型抗生素第一個獲得批準的產品。該類抗生素用于克服早期四環素類抗生素的耐藥性。除了Tigecycline，在過去的40年里，只有兩個新型抗生素上市，分別是輝瑞公司的linezolid (Zyvox)和Cubist公司的daptomycin(Cubicin)。多粘菌素多粘菌素Colistin (polymyxin E)是是混合物多聚氨基酸， 主要針對革蘭氏陰性桿菌 。超級細菌（超級細菌（Super bug）Plasmids Chennai 50 kb to 350 kb； Haryana 118 kb (54%) or 50 kb (36%)； UK isolates 80

5、 kb to greater than 500 kb. Most blaNDM-1 positive plasmids werereadily transferable and prone to rearrangement, losing or (more rarely) gaining DNA on transfer. 質粒的可移動性和可塑性可能意味著在細菌中的廣泛傳質粒的可移動性和可塑性可能意味著在細菌中的廣泛傳播和不斷變異。播和不斷變異。加拿大魁北克省舍布魯克大學附屬醫院的專家日前透露，從加拿大魁北克省舍布魯克大學附屬醫院的專家日前透露，從2003年年初至今，一種普年年初至今，一種普通的

6、腸道細菌通的腸道細菌梭狀芽孢桿菌歷經兩年變異，已成為致命的梭狀芽孢桿菌歷經兩年變異，已成為致命的“超級病菌超級病菌”，它可引，它可引起起65歲以上老年人和服用抗生素的病人產生嚴重痢疾，并最終致死。歲以上老年人和服用抗生素的病人產生嚴重痢疾，并最終致死。該病菌已使這家醫院的該病菌已使這家醫院的100名病人死亡，如不采取緊急行動，這種名病人死亡，如不采取緊急行動，這種“超級病菌超級病菌”很可很可能會引發一場致命傳染病的蔓延。能會引發一場致命傳染病的蔓延。 2010年年9月月5日，日本媒體近日披露，有日，日本媒體近日披露，有46名患者在日本帝京大學醫學部附屬醫院住院期間感染名患者在日本帝京大學醫學部

7、附屬醫院住院期間感染了幾乎可以抵抗所有抗生素的超級細菌了幾乎可以抵抗所有抗生素的超級細菌多重耐藥鮑曼不動桿菌，其中多重耐藥鮑曼不動桿菌，其中9名患者的死亡因感染名患者的死亡因感染此種病菌所致。該醫院曾刻意隱瞞感染事件，導致日本首次出現大規模不動桿菌感染，日本警此種病菌所致。該醫院曾刻意隱瞞感染事件，導致日本首次出現大規模不動桿菌感染，日本警方遂決定傳喚該院醫生，展開調查。方遂決定傳喚該院醫生，展開調查。 經檢測發現，患者感染的不動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經檢測發現，患者感染的不動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這一病菌有效。這一病菌有效。

8、 -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 includin

9、g removal of infected boneA case of resistant S aureus in patientCanada-Edmonton-Webster et al., 2007細菌耐藥機制細菌耐藥機制Drug ImpermeabilityDrug EffluxTarget modificationDrug InactivationOne type of antibiotic can be overcome by different mechanismsDifferent classes of antibiotics can have the same mechanis

10、ms of resistance萬古霉素（萬古霉素（vancomycin ）：由一種鏈霉菌產生的、結構復雜的糖肽類抗生素，專一地抑制）：由一種鏈霉菌產生的、結構復雜的糖肽類抗生素，專一地抑制肽聚糖的生物合成。肽聚糖的生物合成。 萬古霉素通過抑制細菌的生長和繁殖來殺死細菌。這種藥物通過干擾細菌細胞壁結構中的萬古霉素通過抑制細菌的生長和繁殖來殺死細菌。這種藥物通過干擾細菌細胞壁結構中的一種關鍵組分來干擾細胞壁的合成，抑制細胞壁中磷脂和多肽的生成，只是針對革蘭氏陽一種關鍵組分來干擾細胞壁的合成，抑制細胞壁中磷脂和多肽的生成，只是針對革蘭氏陽性菌，對厭氧菌和革蘭氏陰性細菌無效性菌，對厭氧菌和革蘭氏陰性

11、細菌無效 。萬古霉素的藥力較強，且有腎毒性，在其他抗生。萬古霉素的藥力較強，且有腎毒性，在其他抗生素對病菌無效時才會被使用，也就是所謂的最后一線藥物。素對病菌無效時才會被使用，也就是所謂的最后一線藥物。 Howden et al. 2006VISA strains have thicker cell walls-adsorption of Vn by cell-wall D-ala-D-ala residues increased, diffusion decreased-drug is prevented from reaching its active siteVn sensitiveVn

12、 resistantVISA strains have thicker cell wallsThe Case of CMRSA-1CMRSA-1CMRSA-2CMRSA-3CMRSA-4CMRSA-5HungarianNew YorkBrazilianSa 501VF-182Mu3SA MERHIP09662LIM1LY-1999u50SA MER-S20HIP07930HIP09433LIM3BR 5LY-1999 0620-01NRS118HIP1286401020304050agrIaagrIIagrIagrIIIother50250450650850Str

13、ainSAV2095 mRNAexpressionMRSAVISA(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 rRNABussiere et al. Biochemistry 1998Sensitivity Test of 185 Streptococcus pneumoniae IsolatesShen et al. 2008分離自北京兒童的分

14、離自北京兒童的Streptococcus pneumoniae 對大環內酯抗生素的耐藥情況對大環內酯抗生素的耐藥情況erm(B)-carrying elements微生物抗藥機制微生物抗藥機制基因水平轉移基因水平轉移環境中的抗性基因環境中的抗性基因New Resistant BacteriaMutationsXXSusceptible BacteriaResistant BacteriaResistance Gene Transfer基因水平轉移基因水平轉移Horizontal Gene TransferThe movement of genetic material between two

15、organisms. Once incorporated it is then vertically inherited.1- Cyanobacteria -Proteobacteria2- -Proteobacteria Red and brown algae3- -Proteobacteria -Proteobacteria4- -Proteobacteria -ProteobacteriaPan-genome 泛基因組泛基因組The pan-genome includes the “core genome” containing genes present in all strains,

16、 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

17、 variation in gene content among closely related strains). It is a superset of all the genes in all the strains of a species。Transformation- naked DNA, short pieces, common in bacteria that transform .Transduction phage, donor/recipient share receptors, closely related bacteria, DNA: amount in phage

18、 head.Conjugation-plasmids/transposons, cell to cell contact, distant relations, long DNA.Cell death & DNA fragments released into the environmentDNA uptake by related recipient speciesDNA integration with recipients chromosome DNA is either taken up single-stranded or dsDNA is taken up and one

19、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

20、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.Competent cells: able to take up a molecule of DNA via transformation.Only certain strains are competent and this abili

21、ty 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

22、 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.Transformation under natural conditions:Most human /

23、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 pneumoniaeJohnsborg et al. FEMS Microbiol Rev 33，2009可以獲得天然

24、感受態造成了S pneumoniae的多樣性，可利用這一特性進行研究。轉導轉導Transduction: Bacteriophage (phage) mediated gene transferBacterium (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 l

25、yse 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. Generalised transducti

26、on接合ConjugationConjugationCell to cell contact between a donor and a recipient cell in which genes are transferred.微生物耐藥的原因微生物耐藥的原因耐藥靶位點基因突變獲得抗藥基因基因水平轉移（Horizontal gene transfer）可移動遺傳元件（Mobile genetic elements）Hall和和ColhS將整合子定義為，由一系列遺傳元件構成的能夠識別和捕獲移動性基因盒的位點將整合子定義為，由一系列遺傳元件構成的能夠識別和捕獲移動性基因盒的位點特異性重組系統。一

27、個整合子包括編碼整合酶特異性重組系統。一個整合子包括編碼整合酶(inil)的基因及其鄰近的重組識別位點的基因及其鄰近的重組識別位點(attl)。整。整合子不一定包括基因盒，但當基因盒整合入整合子，這些基因盒就成為整合子的一部分。絕合子不一定包括基因盒，但當基因盒整合入整合子，這些基因盒就成為整合子的一部分。絕大多數基因盒本身沒有啟動子序列，基因盒都按照相同的閱讀框方向插入到整合子中，因此大多數基因盒本身沒有啟動子序列，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可以依賴整合子5保守區內的啟動子。保守區內的啟動子。More than 85 different gene cass

28、ettes 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 Rea

29、ctions in integronsIntegron cassette structure and characteristicsStokes, 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 141:3015attC siteICSCS (RYYYAAC)(G / TTRRRY)variable region all contain an attC site (59-base elemen

30、t) - 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 r plTr p m Iinf CL20L35I F 3i n t e g r a s eintI4 orVchintIAMost are 95% identicalcore siteinverse core siteRYYYAACGTTRRRY121-123

31、 nt: VCR126 Kb, 179 cassettes, 3% of genomestomrhAThe V. cholerae superintegronIntegronMazel, D et al (1998) Science 280:605; Heidelberg, Jet al. (2000) Nature 406:477; Rowe-Magnus et al. (1999) Res Mic 150:641微生物抗藥機制微生物抗藥機制基因水平轉移基因水平轉移環境中的抗性基因環境中的抗性基因spore-forming bacteria20 JANUARY 2006 VOL 311 SC

32、IENCE自然環境是抗性基因的儲存庫自然環境是抗性基因的儲存庫Antibiotic-resistance profiling of 75 clonal isolates capable of subsistingon antibiotics.Gautam Dantas, et al. Science 2008Glycopeptide Antibiotic Resistance Genes in Glycopeptide-Producing OrganismsMARSHALL et al. 1998This process demonstrates the importance of bacte

33、rial reservoirs of resistance, including both pathogenic and nonpathogenic organisms. Silbergeld et al. 2008WW, 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藥性的傳播和蔓延抗生素污染加速了耐藥性的傳播和蔓延World Health Org

34、anization 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 promotionAntimicrobials are comple

35、x 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 d

36、estroyed 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.

37、, 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 Tre

38、ated Beef Calves, 2005.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 concentrati

39、on 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 triclos

40、an.* *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, 200

41、6*O.A. Arikan, et.al, Composting Rapidly Reduces Levels of Extractable Oxytetracycline in Manure from Therapeutically Treated Beef Calves, 2005.Katarzyna et al. AEM, 2009Of 9,000 bacterial artificial chromosome clones analyzed, 10 were identified as carrying the known tet(C), tet(W), and tet(40) gen

42、es, as well as novel genes encoding resistance to thetetracyclines minocycline and doxycycline.Tetracyclines and Tetracycline Resistance in Agricultural SoilsSchmitt et al. 2006象山港網箱養殖區沉積物中細菌生態分布象山港網箱養殖區沉積物中細菌生態分布采樣地圖采樣地圖Figure 1 Diversity of the isolates象山港網箱養殖區沉積物中細菌抗藥情況嚴重象山港網箱養殖區沉積物中細菌抗藥情況嚴重Figur

43、e 2 Antibiotics-resistance of the isolates-proteobacteria - proteobacteria Actinobacteria Firmicutes Bacteroidetes分離到的耐藥細菌所含質粒多樣分離到的耐藥細菌所含質粒多樣1 2 3 4 5 6 7 8 9 10 11 12 13 141 D15000 DNA Ladder2,9 Comamonas sp. CNB-13,8 Gemone DNA4 Pseudoalteromonas haloplanktis 5 Exiguobacterium sp. 6 Pseudoclaviba

44、cter helvolus 7 Exiguobacterium aestuarii 10 Roseobacter gallaeciensis11 Roseobacter gallaeciensis12 Brevundimonas nasdae13 Phaeobacter caeruleus14 Labrenzia aggregata Planococcus citreus Bacteria; Firmicutes; Bacillales; Planococcaceae; PlanococcusPlanococcus citreusbacillus subtilisMIC提高了提高了8倍倍Enterococcus Faecom結合轉移結合轉移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 r