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COI Funded Projects: Protists as reservoirs of human pathogens in coastal environments

Project Funded 2008:

Final Report Overview

Human pathogenic bacteria are known to persist in marine environments; however, mechanisms used by these pathogens to survive in seawater and marine sediments are often undetermined. Certain pathogens are capable of surviving within bacterivorous protists – the typical predators of bacteria in aquatic environments. Bacterivorous protists are free‐living, single‐celled eukaryotic microbes (such as ciliates and amoebae). The funding provided by the Coastal Ocean Institute (COI) allowed researchers studying this processes to broaden their investigations of marine bacterivorous protists and their potential roles as reservoirs for human pathogenic bacteria in marine environments.

Funding received from the COI was used to support research conducted by Matt First, an Ocean Life Institute (OLI) Postdoctoral Scholar (9/08‐3/10), Joan Bernhard, and Rebecca Gast. This research focused on determining rates of protist ingestion, digestion, and egestion (the expulsion of intact, intracellular bacteria). These are critical parameters for assessing whether a particular protist is capable of harboring pathogenic bacteria. In summary, this work consisted of culturing bacterivorous protists from coastal environments, including sandy beach sediments, muddy salt‐marsh sediments, and near‐shore waters. Cultured protists were incubated with fluorescently labeled bacteria, and the rates of ingestion, digestion, and egestion were measured via flow cytometry. Additionally, along with a supplement from OLI, funds supported two additional facets to be accomplished: (1) DNA sequencing of cultured protists and bacteria, and (2) ultrastructure analysis of protists incubated with the enteric bacterial pathogen, Campylobacter jejuni. Additionally, the COI funding was used to support travel and presentations of the project. The worked performed, results, and project deliverables are described in more detail below.

Description of work performed

The culturing work performed for this project generated approximately 20 single‐protist cultures of marine bacterivorous protists and 12 bacterial cultures. The funding provided through COI allowed for the sequencing of a subset of these cultures. The alignment of these sequences to those published on public sequence databases allows for the identification of these cultures, providing a permanent record for future researchers. As the prevalence of sequencing increases, many scientific journals require sequence information prior to publication and this information strengthens the overall study.

Campylobacter jejuni is a major source of gastroenteritis in the United States. Campylobacter grows optimally at high temperatures and is sensitive to oxygen – factors that should prevent the persistence of these bacteria in many aquatic environments. Recent evidence indicates that Campylobacter can survive within protists, a trait which can allow their persistence in sub‐optimal environments. We obtained a culture of Campylobacter jejuni to test its survivability and resistance to protist digestion. The bacterivorous protist used for these experiments (Uronema sp.) had several crucial traits necessary for this particular study. Most importantly, this ciliate can grow in brackish waters, therefore, it can be used to test the digestion resistance of Campylobacter across a range of salinities. Populations of the ciliates were incubated with both live Campylobacter and heat‐killed Campylobacter, which was used as a control. Upon completion of the incubation, the co‐cultures were chemically preserved, analyzed via flow cytometry, and critical treatments were selected for ultrastructural analysis using transmission electron microscopy (TEM).

Results and Project Deliverables

Considerable effort was made to validate the use of the flow cytometric method for measuring protist ingestion, digestion, and egestion rates. First, this work established operating procedures for labeling bacteria for detection of intracellular bacteria using flow cytometry. Specifically, we measured whether the fluorescent label was applicable to the various bacteria isolated from the marine environment. Also, we established the length of time that the label was active and determined whether bacteria were able to reproduce after the labeling procedure (a critical factor for intracellular survival).

We also optimized the culture conditions for the protists involved in these experiments and the parameters used for quantifying intracellular bacteria. In order to validate this technique for the measurement of egestion, we employed fluorescently labeled silica micro‐beads (approximately the same size as bacteria). These beads have surface coatings similar to bacterial cell surfaces. Therefore, these beads were ingested and handled similarly to bacteria. However, the indigestible silica beads are eventually egested from the cells. We used these beads to verify that bacterial egestion can be measured using our technique.

Currently, two manuscripts describing this work are in development. The first, which describes and validates the novel flow cytometric method used to measure protists’ ingestion, digestion, and egestion, is being revised to address comments received from the first submission. The second manuscript will include data generated from the study of Campylobacter intracellular survival in the ciliate Uronema sp. under different salinities. The ultrastructural analysis is currently underway ‐ samples have been sectioned and mounted and imaging is expected to occur in June 2010. As an aside, our progress was hampered somewhat by the delay in obtaining clearance to do our work from the WHOI Biosafety Committee. Clearance was not obtained until 12/09, after which First only had 2 months to perform the experiments prior to his departure. Thus, all analyses are not yet completed.

This work has attracted some attention from the scientific media, and an article was recently published online in Oceanus describing this research (link to the article). This article was later highlighted on the Consortium for Ocean Leadership’s website (link to the article). Portions of this work were presented at several venues, including the WHOI Science Made Public lecture series during the summer of 2009 and a meeting of the International Society of Protistologists in 2009. The work will also be presented at the upcoming American Society for Limnology and Oceanography / National Benthological Society joint meeting in June, 2010. In general, the funding provided by COI allowed this project to expand in scope and provided the researchers involved the opportunities to travel and disseminate these results among the scientific community and the interested lay public.

A proposal to expand on this work was submitted to NSF (First, Gast, Bernhard) in 8/09. Although the proposal was declined, it received several positive reviews and constructive comments, which will improve and strengthen future versions of this proposal, although plans for resubmission are indefinite at this time.

Relevant web links:

WHOI Oceanus magazine, posted online October 2, 2009:

http://www.whoi.edu/oceanus/viewArticle.do?id=59746

Consortium for Ocean Leadership, posted online October 28, 2009:

http://www.oceanleadership.org/2009/the-hunt-for-microbial-trojan-horses-should-we-beware-of-protists-bearing-pathogens/

 

Last updated: March 3, 2011