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Rapid detection of predation of Escherichia coli O157:H7 and sorting of bacterivorous Tetrahymena by flow cytometry.

Hernlem BJ, Ravva SV, Sarreal CZ - Front Cell Infect Microbiol (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Foodborne Toxin Detection and Prevention Research Unit, Western Regional Research Center, US Department of Agriculture, Agricultural Research Service Albany, CA, USA.

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.

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Uptake of live vs. heat killed cells of GFP-EcO157 by Tetrahymena monitored over time as the mean and median green fluorescence intensity. Open symbols indicate the case where the E. coli were heat killed prior to feeding. Initial EcO157 concentration was 106 cells mL−1. Predator-prey ratio was 1:100.
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Figure 1: Uptake of live vs. heat killed cells of GFP-EcO157 by Tetrahymena monitored over time as the mean and median green fluorescence intensity. Open symbols indicate the case where the E. coli were heat killed prior to feeding. Initial EcO157 concentration was 106 cells mL−1. Predator-prey ratio was 1:100.

Mentions: The plot in Figure 1 shows a typical time course of fluorescence uptake by Tetrahymena feeding on GFP-EcO157 with an initial concentration of 106 mL−1. Neither heat nor formalin fixation had an adverse effect on GFP fluorescence of the individual E. coli bacteria. Therefore, either method could be used to inactivate the pathogenic bacteria for safety concern when sorting but we chose to use formalin fixation subsequent to feeding so that the bacteria could be fed to Tetrahymena live. In any case, the plot shows a rapid uptake of EcO157 whether live or heat killed, the latter yielding somewhat more peak fluorescence internalization.


Rapid detection of predation of Escherichia coli O157:H7 and sorting of bacterivorous Tetrahymena by flow cytometry.

Hernlem BJ, Ravva SV, Sarreal CZ - Front Cell Infect Microbiol (2014)

Uptake of live vs. heat killed cells of GFP-EcO157 by Tetrahymena monitored over time as the mean and median green fluorescence intensity. Open symbols indicate the case where the E. coli were heat killed prior to feeding. Initial EcO157 concentration was 106 cells mL−1. Predator-prey ratio was 1:100.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4017142&req=5

Figure 1: Uptake of live vs. heat killed cells of GFP-EcO157 by Tetrahymena monitored over time as the mean and median green fluorescence intensity. Open symbols indicate the case where the E. coli were heat killed prior to feeding. Initial EcO157 concentration was 106 cells mL−1. Predator-prey ratio was 1:100.
Mentions: The plot in Figure 1 shows a typical time course of fluorescence uptake by Tetrahymena feeding on GFP-EcO157 with an initial concentration of 106 mL−1. Neither heat nor formalin fixation had an adverse effect on GFP fluorescence of the individual E. coli bacteria. Therefore, either method could be used to inactivate the pathogenic bacteria for safety concern when sorting but we chose to use formalin fixation subsequent to feeding so that the bacteria could be fed to Tetrahymena live. In any case, the plot shows a rapid uptake of EcO157 whether live or heat killed, the latter yielding somewhat more peak fluorescence internalization.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Foodborne Toxin Detection and Prevention Research Unit, Western Regional Research Center, US Department of Agriculture, Agricultural Research Service Albany, CA, USA.

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.

Show MeSH
Related in: MedlinePlus