Abstract
Protozoa are known to harbor bacterial pathogens, alter their survival in the environment and make them hypervirulent. Rapid non-culture based detection methods are required to determine the environmental survival and transport of enteric pathogens from point sources such as dairies and feedlots to food crops grown in proximity. Grazing studies were performed on a soil isolate of Tetrahymena fed green fluorescent protein (GFP) expressing Escherichia coli O157:H7 to determine the suitability of the use of such fluorescent prey bacteria to locate and sort bacterivorous protozoa by flow cytometry. In order to overcome autofluorescence of the target organism and to clearly discern Tetrahymena with ingested prey vs. those without, a ratio of prey to host of at least 100:1 was determined to be preferable. Under these conditions, we successfully sorted the two populations using short 5–45 min exposures of the prey and verified the internalization of E. coli O157:H7 cells in protozoa by confocal microscopy. This technique can be easily adopted for environmental monitoring of rates of enteric pathogen destruction vs. protection in protozoa.
Highlights
Protozoa in the environment have been implicated as both potential hosts harboring pathogens (Barker and Brown, 1994) and as agents enhancing pathogen survival and pathogenicity (Rasmussen et al, 2005; Bichai et al, 2008)
Neither heat nor formalin fixation had an adverse effect on green fluorescent protein (GFP) fluorescence of the individual E. coli bacteria
Either method could be used to inactivate the pathogenic bacteria for safety concern when sorting but FLOW CYTOMETRY AND SORTING The instrument used was a FACSVantage SE flow cytometer (BD Biosciences, San Jose, CA) with an Enterprise II, water-cooled Argon laser (Coherent, Santa Clara, CA)
Summary
Protozoa in the environment have been implicated as both potential hosts harboring pathogens (Barker and Brown, 1994) and as agents enhancing pathogen survival and pathogenicity (Rasmussen et al, 2005; Bichai et al, 2008). Protozoan predation was linked to decreases in EcO157 populations in dairy wastewater (Ravva et al, 2010, 2013) but only three ciliate protozoa were isolated in pure culture (Ravva et al, 2010). For these reasons and difficulties in culturing environmental protozoa, it is important to be able to identify protists in the environment which are actively ingesting bacteria and providing a safe-haven for environmental persistence and transport of EcO157 and other human enteric pathogens. Creative adjustment of signal threshold and detector voltage levels is required when the ratio of protozoa to bacteria is low (Rifa et al, 2002)
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