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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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Histograms showing increasing levels of green fluorescence in Tetrahymena after 1 h of feeding with initial concentrations of GFP-EcO157 from 104 cells mL−1 (top) to 108 cells mL−1 (bottom). Each histogram represents a 10-fold increase in initial concentration.
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Figure 4: Histograms showing increasing levels of green fluorescence in Tetrahymena after 1 h of feeding with initial concentrations of GFP-EcO157 from 104 cells mL−1 (top) to 108 cells mL−1 (bottom). Each histogram represents a 10-fold increase in initial concentration.

Mentions: Indeed, we found that the initial concentration of prey is a vital parameter in determining the maximum uptake of fluorescence. This is clearly shown in Figures 3, 4. At the lower initial concentrations of 104 and 105 bacteria mL−1, the feeding Tetrahymena did not develop enough fluorescence to be clearly discerned from the non-feeding population. On the other hand, as Figure 4 shows, there were very few cells which had no increased fluorescence when the initial bacterial concentrations were 107 and 108 bacteria mL−1. The mean fluorescence of the individual E. coli and unfed Tetrahymena in this experiment were 18 and 16, respectively, on the scale shown.


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)

Histograms showing increasing levels of green fluorescence in Tetrahymena after 1 h of feeding with initial concentrations of GFP-EcO157 from 104 cells mL−1 (top) to 108 cells mL−1 (bottom). Each histogram represents a 10-fold increase in initial concentration.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Histograms showing increasing levels of green fluorescence in Tetrahymena after 1 h of feeding with initial concentrations of GFP-EcO157 from 104 cells mL−1 (top) to 108 cells mL−1 (bottom). Each histogram represents a 10-fold increase in initial concentration.
Mentions: Indeed, we found that the initial concentration of prey is a vital parameter in determining the maximum uptake of fluorescence. This is clearly shown in Figures 3, 4. At the lower initial concentrations of 104 and 105 bacteria mL−1, the feeding Tetrahymena did not develop enough fluorescence to be clearly discerned from the non-feeding population. On the other hand, as Figure 4 shows, there were very few cells which had no increased fluorescence when the initial bacterial concentrations were 107 and 108 bacteria mL−1. The mean fluorescence of the individual E. coli and unfed Tetrahymena in this experiment were 18 and 16, respectively, on the scale shown.

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