Helicobacter Pylori--Nutrition, Serum and Culturability

描述(由申请人提供)：幽门螺杆菌是一种挑剔的微需氧微生物，与消化性溃疡和胃癌密切相关。这种微生物的生长要求只有部分确定，从体内部位和环境中分离幽门螺杆菌仍然具有挑战性。幽门螺杆菌通常需要血液或血清才能在体外生长。我们的初步实验提供了对生长和血清需求的见解，并揭示了通过使用部分定义的培养基而不是标准的复杂培养基来从冷冻原料中回收幽门螺杆菌显著提高。我们假设，使用复杂的培养基会抑制休眠或氧化应激生物体的恢复，从而排除从环境来源分离幽门螺杆菌。我们建议通过进一步定义生长需求和确定血清中最负责生长促进的成分(S)来扩展我们的初步研究。将筛选幽门螺杆菌突变体转座子文库，以识别在没有血清的特定介质中不能生长的克隆。由于血清既能促进生长，又能延长培养活力，本筛选中发现的基因有望为血清在幽门螺杆菌生长中的作用提供线索。这些基因还可能在幽门螺杆菌的稳定期生存中发挥作用，并可能影响生物体的毒力和传播。优化的培养基将与其他活性测量结合使用，以评估幽门螺杆菌从肠道、唾液和粪便等部位的胃外存活情况。这些研究的结果应该会扩大对幽门螺杆菌生长要求和相关基因的理解，特别是与生物体从各种环境条件中恢复有关的要求。这项研究将为临床和研究应用提供更有用的培养基配方，也可能适用于其他挑剔细菌的培养。

DESCRIPTION (provided by the applicant): Helicobacter pylori is a fastidious, microaerophilic organism strongly associated with peptic ulcers and gastric carcinoma. Growth requirements of this organism have only been partially defined, and isolation of H. pylori from in vivo sites and the environment remains challenging. H. pylori normally requires blood or serum for growth in vitro. Our preliminary experiments provide insights into growth and serum requirements and reveal that recovery of H. pylori from frozen stocks is significantly improved through the use of a partially defined medium rather than standard complex media. We hypothesize that the use of complex media inhibits recovery of dormant or oxidatively stressed organisms, precluding isolation of H. pylori from environmental sources. We propose to extend our preliminary studies by further defining growth requirements and identifying the component(s) of serum most responsible for growth augmentation. A transposon library of H. pylori mutants will be screened to identify clones which fail to grow in defined medium without serum. Since serum both augments growth and prolongs culture viability, genes identified in this screen are expected to provide clues regarding serum's function in H. pylori growth. These genes may also play a role in stationary phase survival of H. pylori and may impact virulence and transmission of the organism as well. The optimized medium will be used in conjunction with other measurements of viability to evaluate extragastric survival of H. pylori from sites such as the intestine, saliva, and feces. Results of these studies should extend the understanding of H. pylori growth requirements and the genes involved, particularly requirements relevant to recovery of the organism from various environmental conditions. This research will provide more useful media formulations for both clinical and research applications, and may also be applicable to the culture of other fastidious bacteria.