Department of Biology, Drexel University, 3141 Chestnut St., Philadelphia, PA 19104, USA.
Department of Biology, Drexel University, 3141 Chestnut St., Philadelphia, PA 19104, USA; Department of Nutrition Sciences, Drexel University, Mail Stop 1030, 245 N. 15th St., Philadelphia, PA 19102, USA.
Exp Parasitol. 2014 May;140:39-43. doi: 10.1016/j.exppara.2014.03.012. Epub 2014 Mar 13.
Campylobacter jejuni is one of the leading causes of diarrheal illness worldwide. It is persistent in the environment and on poultry despite its microaerophilic nature and sensitivity to dessication and pH. Studies have demonstrated that C. jejuni co-incubated with Acanthamoeba spp. may be protected from harmful environmental factors. Research in this area, however has included a range of different methodologies for co-incubation, recovery of bacteria and amoebae, and verification of internalization. In this study a modified gentamicin protection assay (mGPA) was developed with a standardized co-incubation procedure. The mGPA addresses limitations of the traditional GPA by providing quantification of the rate of internalization, or lack of internalization, of C. jejuni by Acanthamoeba castellanii. The mGPA described here utilizes tubes instead of cell culture plates allowing for determination of exact numbers of A. castellanii and C. jejuni to be co-incubated prior to addition to tubes. Additionally, the mGPA allows for the incorporation of C. jejuni-only controls to determine the fate of C. jejuni throughout the assay in the absence of A. castellanii. Using the mGPA it was determined that on average 1.6×10(5) C. jejuni (or 0.006% of initial 1×10(9) inoculum) survive the assay in the absence of A. castellanii. Additionally, results obtained with the mGPA demonstrated that while co-incubation with amoebae sometimes (56% of co-incubations) provided a protective effect for C. jejuni, in other cases it did not provide any protective effect (39% of co-incubations), and in at least one case there was a statistically significant higher recovery of C. jejuni in controls when compared to C. jejuni co-incubated with amoebae. The modified gentamicin protection assay described here allows better quantification of the rate and incidence of internalization of bacteria by amoebae. Use of the standardized mGPA developed here with varying environmental parameters and/or strains of bacteria and amoebae may provide insight into factors which are involved in the initiation of internalization of bacteria by amoebae.
空肠弯曲菌是世界范围内导致腹泻疾病的主要原因之一。尽管它是微需氧的，对干燥和 pH 值敏感，但它在环境中和家禽中仍然存在。研究表明，与棘阿米巴属共同孵育的空肠弯曲菌可能会免受有害环境因素的影响。然而，该领域的研究包括了一系列不同的共同孵育方法、细菌和变形虫的回收以及内化的验证。在这项研究中，开发了一种改良的庆大霉素保护测定法（mGPA），并采用了标准化的共同孵育程序。mGPA 通过提供空肠弯曲菌被棘阿米巴属内化的速率或缺乏内化的定量来解决传统 GPA 的局限性。这里描述的 mGPA 使用试管而不是细胞培养板，允许在添加到试管之前确定要共同孵育的棘阿米巴属和空肠弯曲菌的确切数量。此外，mGPA 允许加入仅含有空肠弯曲菌的对照物，以确定在没有棘阿米巴属的情况下整个测定过程中空肠弯曲菌的命运。使用 mGPA 确定，在没有棘阿米巴属的情况下，平均有 1.6×10(5)个空肠弯曲菌（或初始 1×10(9)接种物的 0.006%）在测定中存活。此外，mGPA 的结果表明，尽管与变形虫共同孵育有时（56%的共同孵育）为空肠弯曲菌提供了保护作用，但在其他情况下则没有提供任何保护作用（39%的共同孵育），并且在至少一个情况下，与变形虫共同孵育的空肠弯曲菌对照物相比，空肠弯曲菌的回收率有统计学意义上的显著增加。这里描述的改良庆大霉素保护测定法允许更好地定量变形虫内化细菌的速率和发生率。使用这里开发的标准化 mGPA 并结合不同的环境参数和/或细菌和变形虫菌株，可能会深入了解参与变形虫内化细菌的起始的因素。
