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

Abstract

Campylobacter jejuniis the leading cause of bacterial gastroenteritis worldwide. Transmission to humans occurs through consumption of contaminated food or water. The conditions affecting the persistence of C. jejuniin the environment are poorly understood. Some protozoa package and excrete bacteria into multilamellar bodies (MLBs). Packaged bacteria are protected from deleterious conditions, which increases their survival. We hypothesized that C. jejuni could be packaged under aerobic conditions by the amoeba Acanthamoeba castellanii or the ciliate Tetrahymena pyriformis, both of which are able to package other pathogenic bacteria.A. castellanii did not produce MLBs containing C. jejuni In contrast, when incubated with T. pyriformis,C. jejuni was ingested, packaged in MLBs, and then expelled into the milieu. The viability of the bacteria inside MLBs was confirmed by microscopic analyses. The kinetics of C. jejuni culturability showed that packaging increased the survival of C. jejuniup to 60 h, in contrast to the strong survival defect seen in ciliate-free culture. This study suggests that T. pyriformis may increase the risk of persistence of C. jejuniin the environment and its possible transmission between different reservoirs in food and potable water through packaging.

FIG 1

Interactions of C. jejuni with A. castellanii. Transmission electron micrographs show A. castellanii cocultured with C. jejuni (ratio of 1:1,000) in rich medium. (A) Morphology of vegetative A. castellanii, cultured without C. jejuni. (B) After 3 h, C. jejuni cells (black curved rods) are close to the membrane of the amoeba, and one bacterium is in a phagocytic cup (white arrow). (C) After 3 h, curved-rod bacteria are inside amoebal phagosomes (white arrow). (D) After 24 h of coculture, there is an accumulation of coccoid C. jejuni surrounding A. castellanii. No bacteria are visible inside phagosomes of vegetative A. castellanii.

FIG 2

C. jejuni is packaged in excreted vesicles by T. pyriformis. C. jejuni was cocultured with T. pyriformis for 24 h, at an MOI of 1:1,000 (T. pyriformis/C. jejuni) (A and B) or an MOI of 1:10,000 (C and D), before being fixed and processed for DAPI staining. The average size of the excreted vesicles was 2 to 5 μm. Images of differential interference contrast (DIC) microscopy (A and C) and epifluorescence microscopy (DAPI) (B and D) are shown. The insets in panels C and D show magnification (3.3×) of a typical excreted vesicle containing C. jejuni.

FIG 3

Various multilamellar densities in C. jejuni containing MLBs. C. jejuni was cocultured with T. pyriformis for 24 h before being fixed and processed for TEM. (A) T. pyriformis vegetative cells in fresh PCB. Magnification, ×500. (B) T. pyriformis vegetative cells in fresh PCB. Magnification, ×1,500. (C) Food vacuoles without bacteria in T. pyriformis after 24 h at an MOI of 1:1,000. (D) Food vacuoles of T. pyriformis cocultured with bacteria (at an MOI of 1:10,000), containing dozens of C. jejuni cells well packaged in multilamellar structures (white circles). (E) Food vacuole containing bacteria packaged in a dark ultracompact multilamellar structure (magnification of the top white circle in panel D). (F) Intracellular food vacuole containing bacteria packaged in a clear relaxed multilamellar structure (magnification of the bottom white circle in panel D). (G) Free excreted MLB containing 4 visible bacteria (inset). The increased magnification shows that the coating around the bacteria is quite compact and multilammellar. (H) Dozens of C. jejuni bacteria packaged in a free excreted MLB with a compact multilamellar profile. (I) Dozens of C. jejuni bacteria packaged in a free excreted MLB with a relaxed irregular multilamellar profile.

FIG 4

Packaging of C. jejuni in MLBs maintains its culturability over time. T. pyriformis was cocultured with C. jejuni at an MOI of 1:1,000. At each time point, an aliquot was taken and the numbers of total MLBs and MLBs filled with C. jejuni (packaged C. jejuni) were counted by microscopy (A) and the number of viable C. jejuni present was determined by CFU counting (B). As a control, C. jejuni was incubated in PCB without T. pyriformis. DL, detection limit. Each point is the mean of three separate observations, with error bars representing the standard deviations. We used an unpaired Student t test to assess statistical significance for each time point. *, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005, versus control C. jejuni.

FIG 5

Viability of C. jejuni inside MLBs. A coculture of T. pyriformis and C. jejuni at an MOI of 1:1,000 was incubated at 25°C. At each time point, an aliquot of the coculture was stained with the LIVE/DEAD BacLight solution and images were acquired with a confocal microscope (magnification, 630×) to visualize living (green) and dead (red) C. jejuni cells inside excreted MLBs.