Development of a Modified Gentamicin Protection Assay to Investigate Interactions between Campylobacter jejuni and Acanthamoeba castellanii

Campylobacter jejuni is one of the leading causes of bacterial foodborne illness in the U.S. and the leading bacterial cause of diarrheal illness worldwide. C. jejuni is persistent on poultry products despite its sensitivity to many environmental factors. Protozoa have been studied for their involvement in the internalization and subsequent protection of bacterial pathogens including C. jejuni. In the work presented here a modified gentamicin protection assay (mGPA) was developed to further investigate the internalization, and/or protection of C. jejuni using the amoebal species Acanthamoeba castellanii. This mGPA utilizes tubes instead of the commonly used cell culture plates. Using tubes allows for the inclusion of a C. jejuni only control to quantify C. jejuni survival through the assay independent of the presence of amoebae. Additionally by not working with an amoebal monolayer, this assay is able to provide a more consistent amoeba inoculum and more stable washing step. Previous studies employing a traditional GPA determined internalization by the recovery of C. jejuni at the conclusion of the assay. This mGPA determines internalization by the recovery of higher levels of C. jejuni in the presence of amoebae compared to C. jejuni controls in the absence of amoebae. Using this mGPA, it was determined that C. jejuni NCTC 11168 does survive the assay independent of the presence of amoebae. Taking into account this C. jejuni survival, it was determined that internalization of C. jejuni by amoebae occurs but not consistently. Further investigation provided evidence that internalization may be strain specific and that possible virulence factors may be involved in the occurrence and extent of internalization. Finally the sensitivity of A. castellanii to antimicrobial compounds commonly used in poultry processing was investigated. Results indicated that A. castellanii is susceptible to these antimicrobials at concentrations acceptable for use in poultry processing; however their resistance is higher than C. jejuni. This resistance increases with the addition and increase of organic load, supporting the potential for amoebae to provide a protection to C. jejuni. Overall A. castellanii has shown the ability to provide a protective effect to C. jejuni and may aid in environmental survival.