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Summary
Abstract
Introduction
Protocol
Résultats
Discussion
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Immunologie et infection

新的细胞内病原体由Amoebal共培养和Amoebal富集途径探索

Published: October 27, 2013
doi:
10.3791/51055
, , ,
1Institute of Microbiology,University Hospital Center and University of Lausanne

Summary

Amoebal共存是使用贴壁变形虫以选择性地生长细胞内的病原体能够抵抗吞噬细胞如变形虫和巨噬细胞的细胞培养系统。因此，它代表发现新的传染性病原体的关键工具。 Amoebal富集允许发现其特定的细胞内细菌的新amoebal品种和。

Abstract

细胞内病原体如军团菌，分枝杆菌和衣原体样生物是难以分离，因为它们往往较差或根本不生长在所有上，通常是用于培养细菌的选择性培养基。由于这个原因，许多这些病原体被发现仅在最近或以下重要的爆发。这些病原体往往与变形虫，它作为宿主细胞，并允许细菌的存活和生长有关。我们打​​算在这里提供了两种技术，使孤立的细胞内病原体和表征目前临床或环境样品中的示范：在amoebal共培养和amoebal富集。 Amoebal共培养使细胞内细菌的恢复由所研究的样品接种到一个amoebal草坪，可感染并裂解由存在于样品中的胞内细菌。 Amoebal富集使得目前临床或环境样品中变形虫的恢复。钍是可能导致发现新的amoebal物种，但也是新的细胞内细菌在这些变形虫专门生长。总之，这两个技术有助于发现能够在变形虫长出新的细胞内细菌。因为他们的感染变形虫和抵制吞噬能力，​​这些细胞内的细菌也可能会被巨噬细胞吞噬逃脱，因此，是致病性高等真核生物。

Introduction

分子诊断来临之前，目前在环境壁龛或临床样品中的微生物经培养他们在不同的选择性培养基，主要是在琼脂培养皿中经常发现。细菌菌落和其代谢活性的表现型，让细菌分类在物种水平。肉汤也可以被用于增加检测的灵敏度。然而，这两种技术不允许该生长缓慢或根本没有在这些介质上的细菌的恢复。这就是为什么分子生物学方法被如此广泛地采用了时下的原因。然而，检测DNA提供了对细菌的生存能力没有线索。另外，相反地，以培养，分子生物学方法不会导致菌株，可以进一步表征。

研究病原体生长不良固体培养基上，或需要细胞生长是复杂的。大多数这些“难以生长”的细菌都讲究不及物动词脱细胞的细菌，经常发现其特征如下，因为它是为嗜肺军团菌的情况下大规模爆发。这种细菌的特点如下，一位美国退伍军人大会期间发生的爆发。多达182人被感染，29死于严重肺炎1,2。后来证明，变形虫是这种细菌的自然宿主，而他们在酒店空调系统和供水网络的存在是在所谓的退伍军人症3爆发的起源。

变形虫是目前全球范围内的土壤，空气，水和人类志愿者（4审查）的鼻腔粘膜分离。这些“自由生活”变形虫一般都划分在自主的环境，但有时可能会侵入许可主机5。通过吞噬作用和随后的溶酶体消化HY上的各种微生物饲料阿米巴drolases 6。许多兼性或专性细胞内细菌能够抵抗消化，从而感染和阿米巴划分为例如军团菌 ， 衣原体相关的细菌或分枝杆菌（7审阅和8）。自由生活的阿米巴可能是一个重要的潜在水库已尚未被发现的细胞内细菌。这导致本集团在洛桑实现两个主要的技术，称为amoebal共培养和amoebal富集，这使得不同的群体从不同环境样品9-15隔离了几个新的专性细胞内微生物。

由于变形虫是专业放牧吞噬细胞对细菌，能抵抗吞噬和这些原生生物生长里面的细菌也可能拓殖人的吞噬细胞和致病走向人类。这是部分地显示出对某些衣原体相关的细菌，如Waddlia町ndrophila。 W. chondrophila不仅可以在变形虫而且在一些细胞类型中，如哺乳动物上皮细胞，巨噬细胞，和鱼细胞系16-18成长。该amoebal共培养也出现相关检测细胞内细菌的临床标本19,20，包括大便这是严重污染，不同的细菌种类21。

在这里，我们描述amoebal共培养和amoebal富集的主要步骤，包括：（一）治疗环境或临床标本;变形虫（二）在无菌介质中生长，对大肠杆菌的细菌草坪及（c）选择和表征胞内细菌。

Protocol

1。 Amoebal共培养 1.1样品制备 环境样品 水样 通过0.22μm孔径的膜过滤水样（500毫升至1升）。然后，摇动膜在页面的阿米巴盐水介质首席助理秘书长（120毫克氯化钠，4毫克硫酸镁4•7H 2 O，4毫克氯化钙2•2H 2 O，142毫克的Na 2 HPO 4，和136毫克的KH 2 PO 4在1升的蒸馏水）。 固体?…

Representative Results

使用amoebal共培养和amoebal富集，环境和/或致病菌的整个范围被发现（ 表1）。 Amoebal共培养采​​用由本集团及其他分析环境样品，水处理厂和配水系统。范围广泛的微生物可能是孤立使用这种技术。分离amoebal共培养中最常见的细菌是分枝杆菌属的成员，可以从污水处理厂，并从水网13,14,24,27,28回收。 军团菌和α-变形细菌物种也可从污水处?…

Discussion

Amoebal共培养和amoebal富集是使许多新的细菌和amoebal物种的隔离有效的方法。用这些方法得到的结果证实了这两种阿米巴变形虫和抗细菌在环境中无处不在，最有趣的是在被认为通过化学处理，如氯化，臭氧控制人为供水网络。 Amoebal共培养和amoebal富集是必不可少的工具来隔离和培养这些潜在的病原微生物，并获得菌株纯培养，才能再进一步研究其生物学和致病性。最近，这种方法被改编为巨病毒…

Divulgations

The authors have nothing to disclose.

Acknowledgements

我们感谢箴。伯纳德香格里拉斯科拉的有益的技术咨询和amoebal共培养和amoebal充实有趣的讨论。我们也感谢博士文森特托马斯，他在实施该技术在我们实验室的帮助。

Materials

Name of Reagent/ Equipment Company Catalog Number Comments/Description
Glucose monohydrate Merck, Darmstadt, Germany 108342
0.22 μm pore size membrane Merck Millipore, Darmstadt, Germany SCVPU11RE
proteose peptone Becton-Dickinson, Franklin Lakes, NJ 211693
yeast extract Becton-Dickinson, Franklin Lakes, NJ 212750
Cell culture flasks Becton-Dickinson, Franklin Lakes, NJ 353135
Kova slide Hycor, Indianapolis, IN 87144
cell culture microplates Corning Inc, Corning, NY 3524
Diff-Quik staining kit Siemens Healthcare diagn., Munich, Germany 130832
Ziehl fuchsin Fluka, St-Louis, MI 21820
basic fuchsin Sigma, St-Louis, MI 857843
Phenol Sigma, St-Louis, MI P1037 Corrosive and mutagenic
malachite green oxalate Fluka, St-Louis, MI 63160
Paraformaldehyde 16% solution Electron Microscopy Sciences, Hatfield, PA 15710
Saponin Sigma, St-Louis, MI 84510
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References

  1. Fraser, D. W., et al. Legionnaires’ disease: description of an epidemic of pneumonia. New Engl. J. Med. 297, 1189-1197 (1977).
  2. McDade, J. E., et al. Legionnaires’ disease: isolation of a bacterium and demonstration of its role in other respiratory disease. New Engl. J. Med. 297, 1197-1203 (1977).
  3. Rowbotham, T. J. Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae. J. Clin. Pathol. 33, 1179-1183 (1980).
  4. Rodriguez-Zaragoza, S. Ecology of free-living amoebae. Crit. Rev. Microbiol. 20, 225-241 (1994).
  5. Booton, G. C., Visvesvara, G. S., Byers, T. J., Kelly, D. J., Fuerst, P. A. Identification and distribution of Acanthamoeba species genotypes associated with nonkeratitis infections. J Clin. Microbiol. 43, 1689-1693 (2005).
  6. Brussow, H. Bacteria between protists and phages: from antipredation strategies to the evolution of pathogenicity. Molecular microbiology. 65, 583-589 (2007).
  7. Greub, G., Raoult, D. Microorganisms resistant to free-living amoebae. Clin. Microbiol. Rev. 17, 413-433 (2004).
  8. Thomas, V., McDonnell, G., Denyer, S. P., Maillard, J. Y. Free-living amoebae and their intracellular pathogenic microorganisms: risks for water quality. FEMS Microbiol Rev. 34, 231-259 (2010).
  9. Birtles, R. J., Rowbotham, T. J., Storey, C., Marrie, T. J., Raoult, D. Chlamydia-like obligate parasite of free-living amoebae. Lancet. 349, 925-926 (1997).
  10. Amann, R., et al. Obligate intracellular bacterial parasites of acanthamoebae related to Chlamydia spp. Appl. Environ. Microbiol. 63, 115-121 (1997).
  11. Birtles, R. J., et al. Candidatus Odyssella thessalonicensis’ gen. nov., sp. nov., an obligate intracellular parasite of Acanthamoeba species. Int. J. Syst. Evol. Microbiol. 50, 63-72 (2000).
  12. Thomas, V., Casson, N., Greub, G. Criblamydia sequanensis, a new intracellular Chlamydiales isolated from Seine river water using amoebal co-culture. Environ. Microbiol. 8, 2125-2135 (2006).
  13. Pagnier, I., Raoult, D., La Scola, B. Isolation and identification of amoeba-resisting bacteria from water in human environment by using an Acanthamoeba polyphaga co-culture procedure. Environ. Microbiol. 10, 1135-1144 (2008).
  14. Thomas, V., Loret, J. F., Jousset, M., Greub, G. Biodiversity of amoebae and amoebae-resisting bacteria in a drinking water treatment plant. Environ. Microbiol. 10, 2728-2745 (2008).
  15. Corsaro, D., et al. Novel Chlamydiales strains isolated from a water treatment plant. Environ. Microbiol. 11, 188-200 (2009).
  16. Goy, G., Croxatto, A., Greub, G. Waddlia chondrophila enters and multiplies within human macrophages. Microbes Infect. 10, 556-562 (2008).
  17. Kebbi-Beghdadi, C., Cisse, O., Greub, G. Permissivity of Vero cells, human pneumocytes and human endometrial cells to Waddlia chondrophila. Microbes Infect. 13, 566-574 (2011).
  18. Kebbi-Beghdadi, C., Batista, C., Greub, G. Permissivity of fish cell lines to three Chlamydia-related bacteria: Waddlia chondrophila, Estrella lausannensis and Parachlamydia acanthamoebae. FEMS Immunol. Med. Microbiol. 63, 339-345 (2011).
  19. Fry, N. K., Rowbotham, T. J., Saunders, N. A., Embley, T. M. Direct amplification and sequencing of the 16S ribosomal DNA of an intracellular Legionella species recovered by amoebal enrichment from the sputum of a patient with pneumonia. FEMS Microbiol. Lett. 67, 165-168 (1991).
  20. Rowbotham, T. J. Isolation of Legionella pneumophila serogroup 1 from human feces with use of amebic cocultures. Clin. Infect. Dis. 26, 502-503 (1998).
  21. Greub, G., La Scola, B., Raoult, D. Amoebae-resisting bacteria isolated from human nasal swabs by amoebal coculture. Emerging Infect. Dis. 10, 470-477 (2004).
  22. Isenberg, H. D. . Clinical microbiology procedures handbook. , (1992).
  23. Gimenez, D. F. Staining Rickettsiae in Yolk-Sac Cultures. Stain Technol. 39, 135-140 (1964).
  24. Thomas, V., Herrera-Rimann, K., Blanc, D. S., Greub, G. Biodiversity of amoebae and amoeba-resisting bacteria in a hospital water network. Appl. Environ. Microbiol. 72, 2428-2438 (2006).
  25. Miyamoto, H., et al. Development of a new seminested PCR method for detection of Legionella species and its application to surveillance of legionellae in hospital cooling tower water. Appl. Environ. Microbiol. 63, 2489-2494 (1997).
  26. Lienard, J., et al. Development of a new chlamydiales-specific real-time PCR and its application to respiratory clinical samples. J. Clin. Microbiol. 49, 2637-2642 (2011).
  27. Wang, Y., Ogawa, M., Fukuda, K., Miyamoto, H., Taniguchi, H. Isolation and identification of mycobacteria from soils at an illegal dumping site and landfills in Japan. Microbiol. Immunol. 50, 513-524 (2006).
  28. Corsaro, D., Pages, G. S., Catalan, V., Loret, J. F., Greub, G. Biodiversity of amoebae and amoeba-associated bacteria in water treatment plants. Int. J. Hygiene Environ. Health. 213, 158-166 (2010).
  29. La Scola, B., et al. Legionella drancourtii sp. nov., a strictly intracellular amoebal pathogen. Int. J. Syst. Evol. Microbiol. 54, 699-703 (2004).
  30. Thomas, V., Casson, N., Greub, G. New Afipia and Bosea strains isolated from various water sources by amoebal co-culture. Syst. Appl. Microbiol. 30, 572-579 (2007).
  31. La Scola, B., et al. Amoeba-resisting bacteria and ventilator-associated pneumonia. Emerging Infect. Dis. 9, 815-821 (2003).
  32. Collingro, A., et al. Recovery of an environmental Chlamydia strain from activated sludge by co-cultivation with Acanthamoeba sp. Microbiology. 151, 301-309 (2005).
  33. Lienard, J., Croxatto, A., Prod’hom, G., Greub, G. Estrella lausannensis, a new star in the Chlamydiales order. Microbes Infect. 13, 1232-1241 (2011).
  34. La Scola, B., et al. A giant virus in amoebae. Science. 299, 2033 (2003).
  35. Boyer, M., et al. Giant Marseillevirus highlights the role of amoebae as a melting pot in emergence of chimeric microorganisms. Proc. Natl. Acad. Sci. U.S.A. 106, 21848-21853 (2009).
  36. Thomas, V., et al. Lausannevirus, a giant amoebal virus encoding histone doublets. Environ. Microbiol. 13, 1454-1466 (2011).
  37. Raoult, D., Renesto, P., Brouqui, P. Laboratory infection of a technician by mimivirus. Ann. Internal Med. 144, 702-703 (2006).
  38. Greub, G. Parachlamydia acanthamoebae, an emerging agent of pneumonia. Clin. Microbiol. Infect. 15, 18-28 (2009).
  39. Lamoth, F., Greub, G. Amoebal pathogens as emerging causal agents of pneumonia. FEMS Microbiol. Rev. 34, 260-280 (2010).
  40. Lienard, J. G., Ashbolt, K., Sen, N. J. Ch. 6. Environmental microbiology, current technology and water applications. , 143-162 (2011).
  41. Boughalmi, M., et al. High-throughput isolation of giant viruses of the Mimiviridae and Marseilleviridae families in the Tunisian environment. Environ. Microbiol. , (2012).
  42. Ovrutsky, A. R., et al. Cooccurrence of Free-Living Amoebae and Nontuberculous Mycobacteria in Hospital Water Networks, and Preferential Growth of Mycobacterium avium in Acanthamoeba lenticulata. Appl. Environ. Microbiol. 79, 3185-3192 (2013).

Tags

Keywords: Intracellular PathogensLegionellaMycobacteriaChlamydia-like OrganismsAmoebaeAmoebal CocultureAmoebal EnrichmentIsolationCharacterizationClinical SamplesEnvironmental SamplesBacterial GrowthPhagocytosisMacrophagesPathogenic
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Jacquier, N., Aeby, S., Lienard, J., Greub, G. Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches. J. Vis. Exp. (80), e51055, doi:10.3791/51055 (2013).
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