Acanthamoeba castellanii of the T4 genotype is a potential environmental host for Enterobacter aerogenes and Aeromonas hydrophila

Abstract

Background: Acanthamoeba can interact with a wide range of microorganisms such as viruses, algae, yeasts, protists and bacteria including Legionella pneumophila, Pseudomonas aeruginosa, Vibrio cholerae, Helicobacter pylori, Listeria monocytogenes, Mycobacterium spp., and Escherichia coli. In this capacity, Acanthamoeba has been suggested as a vector in the transmission of bacterial pathogens to the susceptible hosts.

Methods: Here, we used a keratitis isolate of A. castellanii of the T4 genotype and studied its interactions with two bacterial genera which have not been tested before, Enterobacter aerogenes, and Aeromonas hydrophila, as well as E. coli. Assays were performed to determine bacterial association with and invasion of A. castellanii. Additionally, bacterial survival intracellular of A. castellanii trophozoites as well as cysts was determined.

Results: All three bacterial isolates tested, associated, invaded, and survived inside A. castellanii trophozoites as well as A. castellanii cysts. However, E. aerogenes and E. coli exhibited significantly reduced association with and invasion of A. castellanii as compared with A. hydrophila (P < 0.01 using paired T-test, one tail distribution). In the long term survival assays, all three bacterial isolates tested remained viable inside A. castellanii trophozoites, while amoeba remained intact; however A. hydrophila exhibited higher survival inside amoebae (14.54 ± 3.3 bacteria:amoeba ratio) compared with E. aerogenes (3.96 ± 0.7 bacteria:amoeba ratio) and E. coli (5.85 ± 1.1 bacteria:amoeba ratio). A. hydrophila, E. coli, and E. aerogenes remained viable during the encystment process and exhibited higher levels of recovery from mature cysts (14.13 ± 0.89 A. hydrophila:amoeba ratio, 10.13 ± 1.17 E. aerogenes:amoeba ratio, and 11.95 ± 0.7 E. coli:amoeba ratio).

Conclusions: A. hydrophila and E. aerogenes also joined the ranks of other bacteria that could benefit from A. castellanii. Because cysts can be airborne, these findings suggest that Acanthamoeba is a potential vector in the transmission of A. hydrophila and E. aerogenes to susceptible hosts.

Figure 1

Acanthamoeba castellanii exhibited reduced association with Enterobacter aerogenes compared with Aeromonas hydrophila. Association assays were performed to determine bacterial association with A. castellanii as described in “Methods”. (A) represents ratio of bacteria per amoeba, while (B) represents bacteria associated with amoeba (% of the original inoculum). Results are represented as the mean ± standard error of three independent experiments performed in duplicate.

Figure 2

Enterobacter aerogenes and Aeromonas hydrophila that invaded and/or were taken up by Acanthamoeba castellanii. Assays were performed to determine bacterial invasion into or phagocytosis by A. castellanii as described in “Methods”. (A) represents ratio of bacteria per amoeba, while (B) represents bacteria associated with amoeba (% of the original inoculum). Results are represented as the mean ± standard error of three independent experiments performed in duplicate.

Figure 3

Enterobacter aerogenes and Aeromonas hydrophila that survived intracellularly from Acanthamoeba castellanii trophozoites. Assays were performed to determine bacterial survival inside A. castellanii as described in “Methods”. (A) represents ratio of bacteria per amoeba, while (B) represents bacteria associated with amoeba (% of the original inoculum). Results are represented as the mean ± standard error of three independent experiments performed in duplicate.

Figure 4

Enterobacter aerogenes and Aeromonas hydrophila encystment process of Acanthamoeba castellanii and recovery from mature cysts. Assays were performed to determine bacterial survival inside A. castellanii during encystment and their recovery from mature cysts as described in “Methods”. (A) represents ratio of bacteria per amoeba, while (B) represents bacteria associated with amoeba (% of the original inoculum). Results are represented as the mean ± standard error of three independent experiments performed in duplicate. (C) representative micrograph of A. castellanii cysts containing Escehrichia coli K1, Enterobacter aerogenes and Aeromonas hydrophila. Images were taken using light microscopy under different magnifications (200×, 400×, 1000×). Arrows indicate bacteria.

Figure 5

Representative micrograph of Acanthamoeba castellanii interactions with Escehrichia coli K1, Enterobacter aerogenes and Aeromonas hydrophila. Assays were performed to determine bacterial association with and invasion of A. castellanii, as well as bacterial survival inside A. castellanii cysts as described in “Methods”. Finally, samples were stained using Gram staining and observed under a microscope (×400). Note that extracellular bacteria are observed in association assays only, suggesting that gentamicin is effective in killing extracellular bacteria in both invasion as well as intracellular survival assays. Arrows indicate bacteria.