一种蜱源性耐盐耐碱菌株Oceanobacillus oncorhynchi IMH的首次分离与鉴定
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摘要
【目的】本试验以源自呼伦贝尔地区的优势蜱种草原革蜱为材料,从其体内分离得到可疑细菌,应用常规方法和分子生物学方法对该菌株进行分离及鉴定,并对其致病性进行探索,为内蒙古地区蜱虫生物多样性和环境相关性研究奠定了基础。【方法】首先,通过形态学观察,生理生化试验、药物敏感试验以及16S r RNA序列分析,并构建系统发育树,确定该菌株的分类地位;其次对昆明小鼠进行致病性实验。【结果】该菌株革兰氏染色呈G+杆菌,在高盐培养基(5%Na Cl)和p H为9的碱性培养基中生长良好;该菌对不同糖类发酵能力非常有限,只利用部分糖类(如西蒙氏枸橼酸盐试验结果为阳性)产气,但是不产酸;对个别化学药(如环丙沙星)和多数抗生素(如复方新诺明、庆大霉素、红霉素及头孢噻肟等)显示为敏感,而对抗生素卡那霉素和阿莫西林表现为耐药性;经16S r RNA基因序列和系统发育树分析发现,该菌株与GenBank数据库中已注册的多株Oceanobacillus oncorhynchi的同源性为99%以上;该菌在血平板溶血试验中呈现出不完全α溶血,而感染昆明小鼠,观察7 d无明显致病性。【结论】本试验首次分离鉴定了一株蜱源性O.oncorhynchi,并对上述菌株重新命名为O.oncorhynchi IMH,并在Gen Bank中进行注册(LC213016.1),更加丰富了Gen Bank中相关数据。
[Objective] In this study, an unknown bacterium was isolated from Dermacentor nuttalli-a dominant tick species in the area of Hulunbuir. Then we identified the bacterium by molecular biology. We also explored its pathogenicity. [Methods] First, through conventional morphological observation, physiological and biochemical analysis, drug sensitivity test, 16 S rRNA gene sequence analysis and constructing phylogenetic tree, we confirmed the taxonomic status of the bacterium. Then we did pathogenicity experiment on mice. [Results] This bacterium was Gram-positive and grew well in high-salt medium(5% Na Cl) and alkaline medium(pH 9), but its ability was limited to ferment sugar, instead it only used some sugar(Simon's citrate test showed positive results) to make aerogenesis but not acid. It is sensitive for individual chemicals like ciprofloxacin, and most antibiotics like neomycin, gentamicin, erythromycin and cefotaxime, whereas it was resistance to kanamycin and amoxicillin. From the analysis of the 16 S rRNA gene sequence and the phylogenetic trees, we found that the homology of this bacterial strain and Oceanobacillus oncorhynchi from the Gen Bank database(Gen Bank: GQ121034.1) is over 99%. Besides, this bacterial strain shows incomplete hemolysis and no obvious pathogenicity was observed in Kunming mice 7 days after infection. [Conclusion] A tick-borne O. ncorhynchi strain was isolated, identified and named O. oncorhynchi IMH and registered in Genbank(No. LC213016.1), which enriched the relevant data in Gen Bank.
[Objective] In this study, an unknown bacterium was isolated from Dermacentor nuttalli-a dominant tick species in the area of Hulunbuir. Then we identified the bacterium by molecular biology. We also explored its pathogenicity. [Methods] First, through conventional morphological observation, physiological and biochemical analysis, drug sensitivity test, 16 S rRNA gene sequence analysis and constructing phylogenetic tree, we confirmed the taxonomic status of the bacterium. Then we did pathogenicity experiment on mice. [Results] This bacterium was Gram-positive and grew well in high-salt medium(5% Na Cl) and alkaline medium(pH 9), but its ability was limited to ferment sugar, instead it only used some sugar(Simon's citrate test showed positive results) to make aerogenesis but not acid. It is sensitive for individual chemicals like ciprofloxacin, and most antibiotics like neomycin, gentamicin, erythromycin and cefotaxime, whereas it was resistance to kanamycin and amoxicillin. From the analysis of the 16 S rRNA gene sequence and the phylogenetic trees, we found that the homology of this bacterial strain and Oceanobacillus oncorhynchi from the Gen Bank database(Gen Bank: GQ121034.1) is over 99%. Besides, this bacterial strain shows incomplete hemolysis and no obvious pathogenicity was observed in Kunming mice 7 days after infection. [Conclusion] A tick-borne O. ncorhynchi strain was isolated, identified and named O. oncorhynchi IMH and registered in Genbank(No. LC213016.1), which enriched the relevant data in Gen Bank.
引文
[1]Eisen L.Pathogen transmission in relation to duration of attachment by Ixodes scapularis ticks.Ticks and Tick-borne Diseases,2018,9(3):535-542.
[2]Dantas-Torres F,Chomel BB,Otranto D.Ticks and tick-borne diseases:a one health perspective.Trends in Parasitology,2012,28(10):437-446.
[3]Boldbaatar B,Jiang RR,von Fricken ME,Lkhagvatseren S,Nymadawa P,Baigalmaa B,Wang YW,Anderson BD,Jiang JF,Gray GC.Distribution and molecular characteristics of rickettsiae found in ticks across Central Mongolia.Parasites&Vectors,2017,10:61.
[4]Karnath C,Obiegala A,Speck S,Essbauer S,Derschum H,Scholz H,Kiefer D,Tserennorov D,Dashdavaa O,Tsogbadrakh N,Jigjav B,Pfeffer M.Detection of Babesia venatorum,Anaplasma phagocytophilum and Candidatus Neoehrlichia mikurensis in Ixodes persulcatus ticks from Mongolia.Ticks and Tick-borne Diseases,2016,7(2):357-360.
[5]BarákováI,DerdákováM,SelyemováD,Chvostá?M,?pitalskáE,Rosso F,Collini M,RosàR,Tagliapietra V,Girardi M,Ramponi C,Hauffe HC,Rizzoli A.Tick-borne pathogens and their reservoir hosts in northern Italy.Ticks and Tick-borne Diseases,2018,9(2):164-170.
[6]Ebani VV,Bertelloni F,Mani P.Molecular survey on zoonotic tick-borne bacteria and chlamydiae in feral pigeons(Columba livia domestica).Asian Pacific Journal of Tropical Medicine,2016,9(4):324-327.
[7]Wattanamethanont J,Kaewthamasorn M,Tiawsirisup S.Natural infection of questing ixodid ticks with protozoa and bacteria in Chonburi Province,Thailand.Ticks and Tick-borne Diseases,2018,9(3):749-758.
[8]Cross ST,Kapuscinski ML,Perino J,Maertens BL,Weger-Lucarelli J,Ebel GD,Stenglein MD.Co-infection patterns in individual Ixodes scapularis ticks reveal associations between viral,eukaryotic and bacterial microorganisms.Viruses,2018,10(7):388.
[9]Liu W.Emerging tick borne agents in China.Infectious Disease Information,2017,30(1):11-14.(in Chinese)刘玮.中国新发蜱媒病原体概况.传染病信息,2017,30(1):11-14.
[10]马含笑.真空包装熟肉制品中兼性厌氧污染微生物生物防腐技术的研究.石河子大学硕士学位论文,2011:45-50.
[11]布瑞德.伯杰氏系统细菌学手册.第9版.1986:741-801.
[12]胡桂学,陈金顶,彭远义.兽医微生物学实验教程.第2版.北京:中国农业大学出版社,2015:49-61.
[13]Liu HZ,Yang Y,Xu JL.Analysis of slip agar diffusion method for antimicrobial susceptibility test.The Chinese and Foreign Health Abstract,2013,(12):154-155.(in Chinese)刘海珠,杨燕,徐建利.纸片琼脂扩散法检测抗菌药物敏感试验方法分析.中外健康文摘,2013,(12):154-155.
[14]牛天琦.鸡粪中多重耐药细菌的分离鉴定及介导抗性基因水平转移元件的检测.河南师范大学硕士学位论文,2015:17-38.
[15]Eisen RJ,Eisen L.The blacklegged tick,Ixodes scapularis:an increasing public health concern.Trends in Parasitology,2018,34(4):295-309.
[16]Moming A,Yue XH,Shen S,Chang CC,Wang C,Luo T,Zhang YF,Guo R,Hu ZH,Zhang YJ,Deng F,Sun SR.Prevalence and phylogenetic analysis of Crimean-Congo hemorrhagic fever virus in ticks from different ecosystems in Xinjiang,China.Virologica Sinica,2018,33(1):67-73.
[17]Wang Q,Zhao SS,Wureli H,Xie SS,Chen CF,Wei QQ,Cui BY,Tu CC,Wang YZ.Brucella melitensis and B.abortus in eggs,larvae and engorged females of Dermacentor marginatus.Ticks and Tick-borne Diseases,2018,9(4):1045-1048.
[18]Hubálek Z,Rudolf I.Francisella tularensis prevalence and load in Dermacentor reticulatus ticks in an endemic area in Central Europe.Medical and Veterinary Entomology,2017,31(2):234-239.
[19]Raulf MK,Jordan D,Fingerle V,Strube C.Association of Borrelia and Rickettsia spp.and bacterial loads in Ixodes ricinus ticks.Ticks and Tick-borne Diseases,2018,9(1):18-24.
[20]Bush LM,Vazquez-Pertejo MT.Tick borne illness-Lyme disease.Disease-a-Month,2018,64(5):195-212.
[21]Pukhovskaya NM,Morozova OV,Vysochina NP,Belozerova NB,Bakhmetyeva SV,Zdanovskaya NI,Seligman SJ,Ivanov LI.Tick-borne encephalitis virus in arthropod vectors in the Far East of Russia.Ticks and Tick-borne Diseases,2018,9(4):824-833.
[22]Egyed L,él?P,Sréter-Lancz Z,Széll Z,Balogh Z,Sréter T.Seasonal activity and tick-borne pathogen infection rates of Ixodes ricinus ticks in Hungary.Ticks and Tick-borne Diseases,2012,3(2):90-94.
[23]Sumrandee C,Baimai V,Trinachartvanit W,Ahantarig A.Hepatozoon and Theileria species detected in ticks collected from mammals and snakes in Thailand.Ticks and Tick-borne Diseases,2015,6(3):309-315.
[24]O’Callaghan D,Whatmore AM.Brucella genomics as we enter the multi-genome era.Briefings in Functional Genomics,2011,10(6):334-341.
[25]张琦.四川泡菜中嗜盐微生物的分离与鉴定.西华大学硕士学位论文,2013:24-25.
[2]Dantas-Torres F,Chomel BB,Otranto D.Ticks and tick-borne diseases:a one health perspective.Trends in Parasitology,2012,28(10):437-446.
[3]Boldbaatar B,Jiang RR,von Fricken ME,Lkhagvatseren S,Nymadawa P,Baigalmaa B,Wang YW,Anderson BD,Jiang JF,Gray GC.Distribution and molecular characteristics of rickettsiae found in ticks across Central Mongolia.Parasites&Vectors,2017,10:61.
[4]Karnath C,Obiegala A,Speck S,Essbauer S,Derschum H,Scholz H,Kiefer D,Tserennorov D,Dashdavaa O,Tsogbadrakh N,Jigjav B,Pfeffer M.Detection of Babesia venatorum,Anaplasma phagocytophilum and Candidatus Neoehrlichia mikurensis in Ixodes persulcatus ticks from Mongolia.Ticks and Tick-borne Diseases,2016,7(2):357-360.
[5]BarákováI,DerdákováM,SelyemováD,Chvostá?M,?pitalskáE,Rosso F,Collini M,RosàR,Tagliapietra V,Girardi M,Ramponi C,Hauffe HC,Rizzoli A.Tick-borne pathogens and their reservoir hosts in northern Italy.Ticks and Tick-borne Diseases,2018,9(2):164-170.
[6]Ebani VV,Bertelloni F,Mani P.Molecular survey on zoonotic tick-borne bacteria and chlamydiae in feral pigeons(Columba livia domestica).Asian Pacific Journal of Tropical Medicine,2016,9(4):324-327.
[7]Wattanamethanont J,Kaewthamasorn M,Tiawsirisup S.Natural infection of questing ixodid ticks with protozoa and bacteria in Chonburi Province,Thailand.Ticks and Tick-borne Diseases,2018,9(3):749-758.
[8]Cross ST,Kapuscinski ML,Perino J,Maertens BL,Weger-Lucarelli J,Ebel GD,Stenglein MD.Co-infection patterns in individual Ixodes scapularis ticks reveal associations between viral,eukaryotic and bacterial microorganisms.Viruses,2018,10(7):388.
[9]Liu W.Emerging tick borne agents in China.Infectious Disease Information,2017,30(1):11-14.(in Chinese)刘玮.中国新发蜱媒病原体概况.传染病信息,2017,30(1):11-14.
[10]马含笑.真空包装熟肉制品中兼性厌氧污染微生物生物防腐技术的研究.石河子大学硕士学位论文,2011:45-50.
[11]布瑞德.伯杰氏系统细菌学手册.第9版.1986:741-801.
[12]胡桂学,陈金顶,彭远义.兽医微生物学实验教程.第2版.北京:中国农业大学出版社,2015:49-61.
[13]Liu HZ,Yang Y,Xu JL.Analysis of slip agar diffusion method for antimicrobial susceptibility test.The Chinese and Foreign Health Abstract,2013,(12):154-155.(in Chinese)刘海珠,杨燕,徐建利.纸片琼脂扩散法检测抗菌药物敏感试验方法分析.中外健康文摘,2013,(12):154-155.
[14]牛天琦.鸡粪中多重耐药细菌的分离鉴定及介导抗性基因水平转移元件的检测.河南师范大学硕士学位论文,2015:17-38.
[15]Eisen RJ,Eisen L.The blacklegged tick,Ixodes scapularis:an increasing public health concern.Trends in Parasitology,2018,34(4):295-309.
[16]Moming A,Yue XH,Shen S,Chang CC,Wang C,Luo T,Zhang YF,Guo R,Hu ZH,Zhang YJ,Deng F,Sun SR.Prevalence and phylogenetic analysis of Crimean-Congo hemorrhagic fever virus in ticks from different ecosystems in Xinjiang,China.Virologica Sinica,2018,33(1):67-73.
[17]Wang Q,Zhao SS,Wureli H,Xie SS,Chen CF,Wei QQ,Cui BY,Tu CC,Wang YZ.Brucella melitensis and B.abortus in eggs,larvae and engorged females of Dermacentor marginatus.Ticks and Tick-borne Diseases,2018,9(4):1045-1048.
[18]Hubálek Z,Rudolf I.Francisella tularensis prevalence and load in Dermacentor reticulatus ticks in an endemic area in Central Europe.Medical and Veterinary Entomology,2017,31(2):234-239.
[19]Raulf MK,Jordan D,Fingerle V,Strube C.Association of Borrelia and Rickettsia spp.and bacterial loads in Ixodes ricinus ticks.Ticks and Tick-borne Diseases,2018,9(1):18-24.
[20]Bush LM,Vazquez-Pertejo MT.Tick borne illness-Lyme disease.Disease-a-Month,2018,64(5):195-212.
[21]Pukhovskaya NM,Morozova OV,Vysochina NP,Belozerova NB,Bakhmetyeva SV,Zdanovskaya NI,Seligman SJ,Ivanov LI.Tick-borne encephalitis virus in arthropod vectors in the Far East of Russia.Ticks and Tick-borne Diseases,2018,9(4):824-833.
[22]Egyed L,él?P,Sréter-Lancz Z,Széll Z,Balogh Z,Sréter T.Seasonal activity and tick-borne pathogen infection rates of Ixodes ricinus ticks in Hungary.Ticks and Tick-borne Diseases,2012,3(2):90-94.
[23]Sumrandee C,Baimai V,Trinachartvanit W,Ahantarig A.Hepatozoon and Theileria species detected in ticks collected from mammals and snakes in Thailand.Ticks and Tick-borne Diseases,2015,6(3):309-315.
[24]O’Callaghan D,Whatmore AM.Brucella genomics as we enter the multi-genome era.Briefings in Functional Genomics,2011,10(6):334-341.
[25]张琦.四川泡菜中嗜盐微生物的分离与鉴定.西华大学硕士学位论文,2013:24-25.