|First, Matthew -|
|Park, Nina -|
|Bernhard, Joan -|
|Gast, Rebecca -|
|Hollibaugh, James -|
Submitted to: Journal of Eukaryotic Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 13, 2011
Publication Date: January 5, 2012
Citation: First, M.R., Park, N., Berrang, M.E., Meinersmann, R.J., Bernhard, J.M., Gast, R.J., Hollibaugh, J.T. 2012. Ciliate ingestion and digestion: flow cytometric measurements and regrowth of a digestion-resistant campylobacter jejuni. Journal of Eukaryotic Microbiology. 59(1):12-19. Interpretive Summary: Almost all moist or wet environments occupied by bacteria also harbor single-celled protozoa, many of which feed on bacteria as their major nutrient source. These are called bacterivorous protists and a major class of these organisms is the ciliates. When ciliates are feeding, bacteria are ingested and enclosed in cellular structures that contain digestive enzymes that usually kill and digest the bacteria. However, it is known that some bacteria are capable of resisting digestion, remain alive within the protist and even are protected from harsh environmental conditions. We suspect that Campylobacter jejuni, a food-borne human pathogen, is capable of resisting digestion by some protozoa. This manuscript reports a study in which a method was developed to monitor digestion of bacteria by protists. The bacteria were treated with a special dye that glows, fluoresces, when exposed to certain wavelengths of light if the bacteria are still alive. A flow cytometer is a device that uses a laser of the appropriate wavelength to expose one protist at a time and detects whether there is fluorescence. Using this device, we were able to feed stained bacteria to protists, monitor the ingestion of the bacteria by the protists, and then monitor the killing of the bacteria. We showed that Campylobacter jejuni was able to survive in the protist for a longer duration than a non-pathogenic bacterium. This may indicate that control of protozoa is a requirement for controlling Campylobacter in a food-processing environment.
Technical Abstract: We developed a method to measure ingestion and digestion rates of bacterivorous protists feeding on pathogenic bacteria. We tested this method using the enteric bacteria Campylobacter jejuni and a freshwater colpodid ciliate. Campylobacter and a non-pathogenic bacteria isolated from the environment (Psuedomonas putida) were labeled with a fluorogenic compound (CellTracker Green, CMFDA). This compound fluoresces in intact and active cells, but its fluorescence fades when exposed to low pH environments (such as protist food vacuoles). Ingestion and digestion rates were measured via flow cytometry as the change in ciliate fluorescence over time, which corresponded to the number of intracellular bacteria. The rate of Campylobacter ingestion exceeded the digestion rate. Ciliates retained labeled Campylobacter 5 h after ingestion was stopped. In contrast, ciliates grazing upon bacteria isolated from the environment returned to baseline fluorescence within 5 h, indicating that these bacteria were completely digested. The ability of intracellular Campylobacter to remain viable after ingestion was tested by sorting individual ciliates and bacterial cells into Campylobacter selective media. Campylobacter growth occurred in 20 (±4)% of wells seeded with highly fluorescent ciliates; whereas, only 4 (±1)% of wells seeded with free-living Campylobacter exhibited growth. In addition to determining the frequency of potential protist-pathogen associations in the environment, this method can also be applied to examine the environmental conditions leading to the initiation of protist-pathogen associations.