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1、,Introduction,01,Materials and Methods,02,Results and Discussion,03,CONTENTS,Contents,海绵动物,海绵动物是对一类多孔滤食性生物体的统称t,Microorganisms serve as food particles,which are retained from seawater in the choanocyte chambers（细胞环室）,translocated into the mesohyl（中胶层）interior and digested by phagocytosis（吞噬作用）.Many
2、sponges contain furthermore symbiotic microbial consortia（共生微生物）within their mesohyl matrix that may amount up to nearly half of their biomass（生物量）.,Introduction,Infectious disease is the number one cause of death in tropical countries accounting for approximately half of all fatalities.In addition,
3、infectious disease mortality rates are also increasing in developed countries.,Actinomycetes are of considerable interest owing to their ability to produce new chemical entities with diverse pharmacological activities,Marine actinomycetes in particular have yielded numerous novel secondary metabolit
4、es,Our aim is to isolate and culture actinomycetes from marine sponges and to characterize their potential to produce bioactive compounds,specifically those which inhibit the growth of human pathogens and parasites.,Introduction,Materials and Methods,The first group of sponges(Aplysina aerophoba,Dys
5、idea avara,D.tupha,Hemimycale culumella,Ircinia fasciculata)was collected by SCUBA diving at depths of 320 m in the Mediterranean Sea(Rovinj,Croatia,(GPS:2747.655 N;3412.904 W)in August 2008,The second group(Amphimedon sp.,Callyspongia sp.,C.aff.implexa,Hyrtios erecta,Negombata magnifica,Spheciospon
6、gia vagabunda)was collected at a depth of 10 m in the Red Sea(Ras Mohamed,Sinai,Egypt;(GPS:2747.655 N;3412.904 W)in August 2006,1.Sponge Collection,Materials and Methods,2.Actinomycete Isolation,Sponges were transferred to plastic bags containing seawater and transported to the laboratory.Sponge spe
7、cimens were rinsed in sterile seawater,cut into pieces of ca.1 cm 3,and then thoroughly homogenized in a sterile mortar with 10 volumes of sterile seawater.The supernatant was diluted in ten-fold series(10-1,10-2,10-3)and subsequently plated out on agar plates.,无菌海水中切成1cm3,10体积无菌海水中磨碎,稀释涂布分离,10-1,10
8、-2,10-3,Artificial sea water,Storage,All media were supplemented with 0.2 m pore size filtered cycloheximide（放线菌酮）(100 g/mL),nystatin（制霉菌素）（(25 g/mL)and nalidixic acid（萘啶酸）(25 g/mL)to facilitate the isolation of slow-growing actinobacteria.Cycloheximide and nystatin inhibit fungal growth,while nalid
9、ixic acid inhibits many fast-growing Gram-negative bacteria,Inhibitors,Materials and Methods,2.Actinomycete Isolation,Eight different media M1 19,ISP medium 2 39,Oligotrophic medium(OLIGO)40,M1 plus 14,Actinomycete Isolation Agar(AIA)41,Marine Agar(MA)42,Glycerol Asparagine Agar(GAA)41 and R2A Agar
10、43 were used for the isolation of actinobacteria.,Artificial sea water,Storage,Artificial sea water:(NaCl 234.7 g,MgCl 2.6 H 2 O 106.4 g,Na 2 SO 4 39.2 g,CaCl 2 11.0 g,NaHCO 3 1.92 g,KCl 6.64 g,KBr 0.96 g,H 3 BO 30.26 g,SrCl 2 0,24 g,NaF 0.03 g and ddH 2 O to 10.0 L,Materials and Methods,2.Actinomyc
11、ete Isolation,All media contained Difco Bacto agar(18 g/L)and were prepared in 1 L artificial sea water.To promote the growth of selected sponge-associated actinobacteria,1%“aqueous sponge extract”was added to the autoclaved medium.Aqueous sponge extract was prepared by grinding 20 g of sponge bioma
12、ss in a mortar containing 20 mL of sterile seawater followed by centrifugation(5000 rpm,10 min)and sterilized by filtration through a 0.2 m pore size filter.,Inhibitors,Artificial sea water,Storage,The isolates were maintained on plates for short-term storage and long-term strain collections were se
13、t up in medium supplemented with 30%glycerol at 80 C,Materials and Methods,2.Actinomycete Isolation,The freshly prepared supernatant served as aqueous sponge extract.The inoculated plates were incubated at 30 C for 68 weeks.,Inhibitors,Materials and Methods,3.Molecular Identification and Phylogeneti
14、c Analysis,16S rDNA是细菌的系统分类研究中最有用的和最常用的分子钟，其种类少，含量大（约占细菌RNA含量的80%），分子大小适中，存在于所有的生物中，其进化具有良好的时钟性质，在结构与功能上具有高度的保守性，素有“细菌化石”之称。在大多数原核生物中rDNA都具有多个拷贝，5S、16S、23S rDNA的拷贝数相同。16S rDNA由于大小适中，约1.5Kb左右，既能体现不同菌属之间的差异，又能利用测序技术较容易地得到其序列，故被细菌学家和分类学家接受,基因组的制备,PCR克隆16SRNA,阳性克隆鉴定，测序,同源性分析，系统进化树建立,16S rDNA,Materials a
15、nd Methods,4.Extract Preparation and Anti-infective Activity Screening,基于系统发育的新分支选择出的十四株和基于从属于已知代谢物生产者选择出的六株分别培养在含50ml的5种不同生产培养基(M1,ISP2,OLIGO,AIA and R2A)的100mL锥形烧瓶中。依据它们在30C150rpm摇动下下的生长速率，这种液体培养物生长7到14天。为了使细胞裂解，在持续摇动下向液体培养液中加入一份等体积的甲醇。液体培养基在50ml的离心管中离心（5000 rpm,15 min，室温），将上清液在4 C保存。,盛有放线菌提取物的无菌过
16、滤盘放置在已接种待测病原菌的琼脂板上。在37度（细菌）和30度（酵母）下培养24小时候，抗微生物潜力是通过抑制圈的半径（n=2）潜力估测的。,Materials and Methods,4.Extract Preparation and Anti-infective Activity Screening,抗利什曼原虫活性是沿用Ponte-Sucre等人方法来测试的。简单的说，这涉及到利什曼原虫主要的前鞭毛体在26度，5%co2，96%湿度下存在或不存在提取物时持续24小时的培养的潜伏期。在加入 Alamar-Blue试剂后，板再次培育，在48小时后用elisa实验测定出光学密度。在没化合物条件
17、下的吸光度说明致病菌100%增长。两性霉素B是参考化合物和阳性对照。独立的实验中的每个提取物被重复检测。抗锥虫活性测试是按照Huber和Koella的方法测试的。布氏锥虫布氏菌株TC221在Baltz完全培养基中的培养后，寄生虫的数目暴露在96孔板提取物测试板中。然后将板在5%co2，37度的环境下培养24小时。加Alamar-Blue试剂之后，提取物的活性在48小时后通过MR700 Microplate Reader测试出吸光度来衡量。没有化合物（两性霉素B）的吸光度表明病原体100生长。在两组独立实验中的每个提取物被重复测定。,Results and Discussion,1.Divers
18、ity of Sponge-Associated Actinomycetes,Homogenates from 11 taxonomically different marine sponges collected from Ras Mohamed,Egypt and Rovinj,Croatia were plated on a range of selective media for the isolation of actinomycetes.,Number of actinomycete isolates per sponge species,Results and Discussio
19、n,A comparison of the nearly complete 16S rRNA gene sequences of 52 out of 90 putative actinobacterial strains isolated from these sponges against sequences in the NCBI GenBank database revealed the phylogenetic affiliations to 18 different genera representing 14 families and seven suborders(Supleme
20、ntary Table 1).,Number of actinomycete isolates per actinomycete genera,Results and Discussion,The isolation media of this study were chosen to select for actinobacteria.M1 exhibited the highest recovery producing 27 isolates and MA recovered only two isolates(Figure 1C).,Number of actinomycete isol
21、ates per cultivation media,Results and Discussion,Results and Discussion,Results and Discussion,2.Bioactivity Testing,The antimicrobial activity was assessed by the disc diffusion assay while the anti-parasitic activity was measured in multi-well plates and is represented as the percentage of growth
22、 inhibition in comparison to a reference compound。Of the twenty strains tested,ten produced antimicrobially active metabolites inhibiting at least one of the test pathogens.Five isolates were capable of inhibiting the growth of Gram-positives only,one isolate was active against C.albicans only and o
23、ne isolate showed activity against both groups of pathogens(Table 1).The inhibition was greatest with the Streptomyces sp.RV15 against Staphylococcus aureus.Gram-negative bacteria were not susceptible to our isolates.In terms of the anti-parasitic activity,four isolates exhibited activity against T.
24、brucei and two isolates showed activity against L.major(Table 2).The novel Actinokineospora sp.EG 49 showed highest antitrypanosomal potential at 48%growth inhibition.Anti-parasitic activities from marine-derived actinomycetes are reported only sporadically.For example,a new thiolactone antibiotic,thiolactomycin,from Nocardia strain No.2-200 showed potent antimalarial and antitrypanosomal activities 32.Additionally,the macrolide antibiotic amphotericin B produced by Streptomyces nodosus was used for treatment of leishmaniasis。,Results and Discussion,End,Thankyou,
