Survival characteristics of diarrheagenicEscherichia colipathotypes andHelicobacter pyloriduring passage through the free-living ciliate,Tetrahymenasp.

Abstract

Free-living protozoa have been implicated in the survival and transport of pathogens in the environment, but the relationship between non-Shiga toxin-producing Escherichia coli or Helicobacter pylori and ciliates has not been characterized. Six diarrheagenic pathotypes of E. coli and an isolate of H. pylori were evaluated for their susceptibility to digestion by Tetrahymena, an aquatic ciliate. Tetrahymena strain MB125 was fed E. coli or H. pylori, and the ciliate's egested products examined for viable bacterial pathogens by the BacLight(™) LIVE/DEAD (™) assay, a cell elongation method, and by colony counts. All six diarrheagenic E. coli pathotypes survived digestion, whereas H. pylori was digested. Growth of E. coli on agar plates indicated that the bacteria were able to replicate after passage through the ciliate. Transmission electron micrographs of E. coli cells as intact rods vs. degraded H. pylori cells corroborated these results. Scanning electron microscopy revealed a net-like matrix around intact E. coli cells in fecal pellets. These results suggest a possible role for Tetrahymena and its egested fecal pellets in the dissemination of diarrheagenic E. coli in the environment. This bacterial-protozoan interaction may increase opportunities for transmission of diarrheagenic E. coli to mammalian hosts including humans.

Packaging of Campylobacter jejuni into Multilamellar Bodies by the Ciliate Tetrahymena pyriformis

“…In comparison with the vegetative cells of T. pyriformis, no significant morphological difference in the membrane was noticed after T. pyriformis-C. jejuni coincubation. Our data correlate with previous reports on S. enterica serovar Typhimurium and E. coli, which have no detectable cytotoxic effects on Tetrahymena (33,46,47), in contrast to Acanthamoeba spp., which are killed by other pathogenic bacteria (48)(49)(50)(51).…”

“…The C. jejuni packaged in expelled free MLBs was a little bigger than that in the intracellular vacuoles; the bacteria are mostly rod-shaped, and their size may exceed 2 m, with an average length of 1 m. Here, this phenomenon is probably due to the loosening of MLB membranes after expulsion into the medium, resulting from decreased pressure on the MLBs, compared with that inside the protozoal cell (54). Transmission electron micrographs of E. coli and L. pneumophila contained in MLBs also showed that the bacteria inside food vacuoles of Tetrahymena were smaller than those inside expelled MLBs (47,54).…”

“…As a comparison, the residence times of E. coli and L. pneumophila in Tetrahymena were reported to be approximately 2 to 4 h (47,53,55). It is known that the food vacuoles in ciliates can take as little as 1 h to proceed from ingestion to exocytosis (56).…”

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

“…Additionally, it can take many hours of electron microscope time to acquire enough micrographs to analyze, while many low-power fields can be acquired from cells exposed to fluorescence-based viability dyes, even in the short time afforded for image acquisition. While the provided protocols were optimized with N. gonorrhoeae and primary human neutrophils, they can be adapted to assess the viability of many species of gram-negative and gram-positive bacteria in a variety of cell types, including all phagocytic and nonphagocytic eukaryotic cells 5,[15][16][17][18][19][20][21][22][23][24]26 . Bacterial viability dyes provide a powerful experimental tool to examine mechanisms of bacterial pathogenesis in eukaryotic cells and the ability of eukaryotic cells to control bacterial infection.…”

“…The protocols outlined in this article were used to assess the viability of N. gonorrhoeae that is attached to and inside primary human neutrophils, including in different populations of neutrophil phagosomes 5,13,14 . However, these protocols can be applied to assess viability of gram-positive and gram-negative bacteria in professional phagocytes, nonprofessional phagocytes, and protozoa [15][16][17][18][19][20][21][22][23][24] .…”