一种优化富集技术的隔离<em>节杆菌</em>噬菌体种从土壤样品隔离
Summary
We present an enrichment protocol for the isolation of bacteriophages infecting bacteria in the Arthrobacter genus. This enrichment protocol produces fast and reproducible results for the isolation and amplification of Arthrobacter phages from soil isolates.
Abstract
Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of phages infecting Arthrobacter hosts has been limited, perhaps due to the low success rate of many previous isolation techniques, resulting in an underrepresented group of Arthrobacter phages available for study. The enrichment technique described here, unlike many others, uses a filtered extract free of contaminating bacteria as the base for indicator bacteria growth, Arthrobactersp. KY3901, specifically. By first removing soil bacteria the target phages are not hindered by competition with native soil bacteria present in initial soil samples. This enrichment method has resulted in dozens of unique phages from several different soil types and even produced different types of phages from the same enriched soil sample isolate. The use of this procedure can be expanded to most nutrient rich aerobic media for the isolation of phages in a vast diversity of interesting host bacteria.
Introduction
节杆菌种在土壤环境中无处不在提供能够从这种寄主细菌被噬菌体分离出一个庞大的数量和多样性。在Acintobacteriaceae家族的细菌成员是最显着的降低顽固的化合物，如莠去津和其他各种杀虫剂和除草剂1,2,3他们的代谢途径。虽然大多数研究已经完成使用的节杆菌菌株环境，本属临床分离株血，尿，眼睛，和其他许多人来源的所有显示系统发育不均匀4找到。
虽然有相当广泛的对节杆菌的细菌研究机构，只有少数研究能够感染这种多样化属成员的噬菌体报告。但有趣的是，在此前节杆菌所做的工作噬菌体几个关键的不同的主题接触，如土壤类型<EM>节杆菌物种5，工业用途以减少有害的泡沫在活性污泥处理厂6和工作突出位点特异性重组和整合基因7的目的。
各种富集技术协议已被利用来产生纯的噬菌体分离物在节杆菌物种。早期的程序包括土壤添加了有毒物质尼古丁一样的盐每年超过8引起的时期孵化到能够只感染A的噬菌体球形节杆菌 。研究使用的土壤渗出完成与不稳定的有机物经出现斑块检测技术，生产检测噬菌体，省略了漫长的潜伏期8。但有趣的是，一个类似的技术直接电镀用在过去的几个噬菌体引起同时还具有一个显着低成功率的调查5，理由是低成功率过去的研究<s达> 8。
总体而言，在过去所用的分离技术是值得注意的具有尽管节杆菌属的代表最常见的有氧土壤在实践中很少功效隔离性质的4,9，，范Twest和Kropinski 10本富集方法用于从水和土壤中分离的噬菌体改编自用于充实环境的细菌菌株，但这些富集技术的早期技术被证明效率低下的隔离节杆菌噬菌体。这里所描述的方法的目的是要表明“概念验证”，早期的富集方法可以适于一致地和有效地隔离节杆菌噬菌体，克服了与来自该细菌属中分离的噬菌体相关的先前的技 术挑战。
Protocol
Representative Results
Discussion
尽管许多以前曾试图孤立能够感染主机节杆菌噬菌体的，我们有一点成功使用标准的浓缩过程。细菌富集开发并适于通过面包车Twest和Kropinski 10从环境样品富集的噬菌体的广义方法仍是依据对大多数富集过程。从以往的研究证据表明，直接电镀的方法都对节杆菌菌株的检测斑块尽管有隔离5的成功率极低。与直接镀法，噬菌体是从土壤样品中提取噬菌体缓冲和过滤，以…
Disclosures
The authors have nothing to disclose.
Acknowledgements
资助这项协议的发展是由东南宾夕法尼亚财团高等教育和卡布里尼学院科学系提供。额外的资金和支持来自阿卡迪亚大学和Immaculata大学。我们特别感谢莫文蔚Snetselaar博士在圣约瑟夫大学好心参加我们的孤立的噬菌体电子显微图像。额外的支持是由霍华德·休斯医学研究所科学教育联盟噬菌体猎人推进基因组学和进化科学（SEA-噬菌体）计划提供的。
Materials
| LB Broth powder | Fisher | BP9722-2 | It's best to order these in bulk. |
| Granulated Agar | Fisher | BP1423-2 | It's best to order these in bulk. |
| 0.22 um syringe filters | Fisher | 09-719A | |
| .22 um buchner filters | Fisher | 430320 | More than 50 mL of liquid can be obtained by carefully swapping the receiving tube. |
| Eppendorf Tubes | Fisher | 05-408-129 | |
| 5 mL pipets individual | Fisher | 13-678-11D | |
| 50 mL conical tubes | Fisher | 76002844 | |
| 15 mL conical tubes | Fisher | 76002845 | |
| 10 mL pipets individual | Fisher | 13-676-10J | |
| 25 mL pipets individual | Fisher | 13-676-10K | |
| Whatman qualitative filter paper | Fisher | 1001-824 |
References
- Shapir, N., Mongodin, E. F., Sadowsky, M. J., Daugherty, S. C., Nelson, K. E., Wackett, L. P. Evolution of Catabolic Pathways: Genomic Insights into Microbial s-Triazine Metabolism. J Bacteriol. 189 (3), 674-682 (2007).
- Qingyan, L., Ying, L., Xikun, Z., Baoli, C. Isolation and Characterization of Atrazine Degrading Arthrobacter sp. AD26 and Use of this Strain in Bioremediation of Contaminated Soil. J Environ Sci. 20, 1226-1230 (2008).
- Wang, J., Zhu, L., Liu, A., Ma, T., Wang, Q., Xie, H., Wang, J., Jiang, T., Zhao, T. Isolation and characterization of an Arthrobacter sp. Strain HB-5 that Transforms Atrazine. Environ Geochem Hlth. 33, 259-266 (2011).
- Mages, I. S., Frodl, R., Bernard, K. A., Funke, G. Identities of Arthrobacter spp. And Arthrobacter-Like Bacteria Encountered in Human Clinical Specimens. J Clin Microbiol. 46 (9), 2980-2986 (2008).
- Brown, D. R., Holt, J. G., Pattee, P. A. Isolation and Characterization of Arthrobacter Bacteriophages and Their Application to Phage Typing of Soil Arthrobacters. Appl Environ Microbiol. 35 (1), 185-191 (1978).
- Petrovski, S., Seviour, R. J., Tillett, D. Prevention of Gordonia and Nocardia Stabilized Foam Formation by Using Bacteriophage GTE7. Appl Environ Microbiol. 77 (21), 7864-7867 (2011).
- Le Marrec, C., Moreau, S., Loury, S., Blanco, C., Trautwetter, a. Genetic Characterization of Site-specific Integration Functions of phi AAU2 infecting “Arthrobacter aureus” C70. J Bacteriology. 178 (7), 1996-2004 (1996).
- Casida, L. E., Liu, K. C. Arthrobacter globiformis and Its Bacteriophage in Soil. J Appl Microbiol. 28 (6), 951-959 (1974).
- Einck, K. H., Pattee, P. A., Holt, J. G., Hagedorn, C., Miller, J. A., Berryhill, D. L. Isolation and Characterization of a Bacteriophage of Arthrobacter globiformis. J Virol. 12 (5), 1031-1033 (1973).
- Twest, R., Kropinski, A. M. Bacteriophage Enrichment from Soil and Water. Methods Mol Bio. 501, 15-21 (2009).
- Fullner, K. J., Hatfull, G. F. Mycobacteriophage L5 Infection of Mycobacterium bovis BCG: Implications for Phage Genetics in the Slow-growing Mycobacteria. Mol Microbiol. 26 (4), 755-766 (1997).
- Robb, S. M., Woods, D. R., Robb, F. T. Phage Growth Characteristics on Stationary Phase Achromobacter cells. J Gen Virol. 41 (2), 265-272 (1978).
- Bolger-Munro, M., Cheung, K., Fang, A., Wang, L. T4 Bacteriophage Average Burst Size Varies with Escherichia coli B23 Cell Culture Age. Journal of Experimental Microbiology and Immunology. 17, 115-119 (2013).
