Continuous Real-Time Motility Analysis of Acanthamoeba Reveals Sustained Movement in Absence of Nutrients

Abstract

Acanthamoeba keratitis is a serious ocular infection which is challenging to treat and can lead to blindness. While this pathogen is ubiquitous and can contaminate contact lenses after contact with water, its habits remain elusive. Understanding this organism’s natural behavior will better inform us on how Acanthamoeba colonize contact lens care systems. Acanthamoeba trophozoites were allowed to adhere to either a glass coverslip or non-nutrient agar (NNA) within a flow cell with nutrients (Escherichia coli or an axenic culture medium (AC6)) or without nutrients (Ringer’s solution). Images were taken once every 24 s over 12 h and compiled, and videos were analyzed using ImageJ Trackmate software. Acanthamoeba maintained continuous movement for the entire 12 h period. ATCC 50370 had limited differences between conditions and surfaces throughout the experiment. Nutrient differences had a noticeable impact for ATCC 30461, where E. coli resulted in the highest total distance and speed during the early periods of the experiment but had the lowest total distance and speed by 12 h. The Ringer’s and AC6 conditions were the most similar between strains, while Acanthamoeba in the E. coli and NNA conditions demonstrated significant differences between strains (p < 0.05). These results indicate that quantifiable visual tracking of Acanthamoeba may be a novel and robust method for identifying the movement of Acanthamoeba in relation to contact lens care products. The present study indicates that Acanthamoeba can undertake sustained movement for at least 12 h with and without nutrients, on both rough and smooth surfaces, and that different strains have divergent behavior.

Acanthamoeba spp. aggregate and encyst on contact lens material increasing resistance to disinfection

“…For polystyrene controls and lehfilcon A, there was no indication that any binary fission occurred based on cell count, and eventually individual trophozoites encysted though active trophozoites were visible through the 72 h despite no nutrient source. This supports previous work that shows trophozoites will maintain motility through 24 h with no decrease in activity even without nutrients ( Campolo et al, 2021 ). Here, Acanthamoeba trophozoites show a remarkable response to a surface they identified as inhospitable, responding quickly (<1 h) to many contact lens materials, aggregating and initiating encystment (< 4 h) despite no chemical induction beyond the contact lens surface.…”

“…Additionally, despite being seeded with the same number of cells in the same plate at the same time, the no lens control in most strains tested demonstrated a lower particle count in the field of view than all lenses tested. This was due to amoeba having a larger available space (flat-bottomed well vs. bowl of a contact lens), although they were consistently observed to be evenly dispersed throughout the well both in this study and in previous ones ( Campolo et al, 2021 ). Further, it is a noticeable phenomenon that count increases in all lens materials and in all strains from time 0–0.5 h to 0.5–1.0 h. This is due not only to amoeba settling onto the lens, but also to their inclination to walk down to the bottom of the bowl of the lens upon adherence to the lens, thereby coming into the field of view and being counted.…”

“…To understand the differences in Acanthamoeba behavior on popular lens materials, we investigated six different potentially keratitis-causing strains of Acanthamoeba for 72 h on seven different lens materials, as well as a polystyrene no lens control in which Acanthamoeba appeared to move independently and consistently similar to previously examined surfaces ( Campolo et al, 2021 ; Figures 2 – 5 ; Supplementary Figures S2–S4 ). An experimental timeline was designed to allowed us to observe and quantify aggregation both in the clinically normal periods of when a contact lens might be stored individually in a contact lens case overnight, as well as longer periods to determine if any behavior was transient (de-aggregation of Acanthamoeba on their own without conditions otherwise changing).…”

“…To allow the amoeba to settle onto the lens and compensate for size differences observed between strains, the size of each strain-lens condition was normalized to its own 0.5–1.0 h reference time ( Campolo et al, 2021 ). Additionally, due to the count difference of the no lens control, which was not confined to a smaller field of view by the bowl of the contact lens, the individual particle count of the no lens control was not included in the statistical analysis.…”

Complete Recovery of Acanthamoeba Motility among Surviving Organisms after Contact Lens Care Disinfection

“…Overall, there is a lack of research and understanding regarding Acanthamoeba motility and how it relates to contact lens use, or how it is impacted by CLC disinfection. We recently published a novel method regarding Acanthamoeba motility on control surfaces with and without available nutrients [ 8 ], and found that amoebae were able to remain motile for at least 12 h even in the absence of nutrients [ 9 ]. This indicated that amoebae could potentially travel substantial distances on contaminated lenses, or from a lens to a cornea and into the corneal epithelium, within the normal period of time for either wearing a lens or in which the lens is in its case.…”