Variables Affecting the Recovery of Acanthamoeba Trophozoites

Abstract

While the results of Acanthamoeba testing have been extensively published, laboratories conducting such testing are left to develop their own methods in the absence of a standardized methodology. The wide disparity of methods has resulted in equally inconsistent reported results for contact lens care (CLC) products. This study's objective was to determine the source of these discrepancies by evaluating basic Acanthamoeba biology and their impact on antimicrobial efficacy testing, including the ability of a recovery method to stimulate a single trophozoite to proliferate. Antimicrobial efficacy testing was conducted using well-published Acanthamoeba strains, storage conditions, and growth-based recovery methods. To identify variables that influence results, test solutions with low Acanthamoeba disinfection rates were utilized to prevent differences from being masked by high log reductions. In addition, single-cell proliferation assays were executed to understand the growth requirements to stimulate trophozoite propagation in two recovery methods. These studies indicated that both nutrient density (>10⁶ CFU) and the length of plate incubation (at least 14 days) could significantly influence the accurate recovery of trophozoites. Together, this study emphasizes the need to understand how Acanthamoeba trophozoites biology can impact test methods to create divergent results.

Keywords: Acanthamoeba; contact lens care; efficacy testing; trophozoites.

Figure 1

Strain ATCC 30461 demonstrates a significantly greater log reduction than strain ATCC 50370. Mean ± SE comparison of antimicrobial activity for three test solutions per Acanthamoeba strain as determined on Day 7, 14, and 21 plate reads. Means reflect combined recovery method and source data. a: p < 0.05 vs. 30461 of the same solution and day. n = 36/group.

Figure 2

The source (long-term storage method) of Acanthamoeba has no impact on biocidal efficacy. Mean ± SE of comparison of log reductions for each test solution against (A) Acanthamoeba ATCC 50370 and (B) Acanthamoeba ATCC 30461 trophozoites by long-term maintenance method for Day 7, 14, and 21 plate reads. a: p < 0.05 vs. Solution 1 plug at the same time point, b: p < 0.05 vs. Solution 2 plug at the same time point. n = 6–18/group.

Figure 3

Results show that 96-well recovery changed depending on plate incubation length. Left y-axis: Mean ± SE log reduction of (A) Acanthamoeba ATCC 30461 and (B) Acanthamoeba ATCC 50370 trophozoites for the three test solutions per recovery method as determined on Day 7, 14, and 21 plate reads. Right y-axis: total cells/mL (in log) of the paired inoculum control. a: p < 0.05 vs. 12-well recovery for the same solution at the same day, b: p < 0.05 vs. Day 7 of the same solution and recovery, c: p < 0.05 vs. Day 14 of the same solution and recovery. n = 12–18/group.

Figure 4

Acanthamoeba growth requires at least 14 days to stabilize, with the greatest impact on incubation vs. log reduction results in 96-well method recovery. Left: mean ± SE and associated p-values of log reduction by strain, day, and solution. Right: associated visual trend. Left y-axis: Mean log reduction by day of Acanthamoeba ATCC 30461 and Acanthamoeba ATCC 50370 trophozoites for the three test solutions per recovery method as determined on Day 7, 14, and 21 plate reads. Right y-axis: total cells/mL (in log) of the paired inoculum control. Black lines: 12-well recovery, grey lines: 96-well recovery. n = 12–18/group.

Figure 5

ATCC 30461 recovers faster than ATCC 50370 for both recovery methods, and the 12-well recovery method is the overall fastest recovery method for both strains. Days to final concentration for Acanthamoeba ATCC 30461 and ATCC 50370 trophozoites for inoculum controls, and Solutions 1–3, with all sources/test replicates combined. Days to final concentration were determined as the minimum incubation time for the number of positive wells on a recovery method to stop increasing. Dotted line indicates 14 days, which is the standard incubation time for Acanthamoeba recovery plates [47,48,50]. a: p > 0.05 vs. 96-well of the same strain/solution, b: p > 0.0001 vs. 12-well for ATCC 50370 with same solution, c: p > 0.0001 vs. 96-well for ATCC 50370 for same solution. Checkmarks in the table indicate where plates/replicates had Day of Final concentration after 14 days, resulting in differences in log reduction between Day 14 results and Day 21 results.

Figure 6

The percent viability of a single trophozoite was dependent on strain and E.coli concentration. The number of positive wells following single trophozoite seeding are listed for each strain of Acanthamoeba and the different concentrations of E. coli. The number of seeded wells indicates the total number of possible positive wells. The percent viability of each strain was graphed to demonstrate the impact of E. coli concentration and strain on the recovery of single trophozoites. Black bars: ATCC 50370; grey bars: ATCC 30461. Please see Figure 7 and Figure 8 for statistical analysis of positive wells. NT: Not Tested.

Figure 7

Results show that 96-well recovery and lower E. coli density significantly increase the days to final percent positive rates. Day when individual Acanthamoeba ATCC 50370 trophozoite wells were identified as positive for each recovery method within (A) Inoculum Control and (B) Solution 2 treatment. The percentage of positive wells was calculated using the total number of wells positive by Day 65 as the denominator and the positive wells positive on a particular day as the numerator. a: p < 0.05 vs. Day 7 within the same recovery and E. coli density, b: p < 0.05 vs. Day 14 within the same recovery and E. coli density, c: p < 0.05 vs. 10⁷ CFU/well 96-well, d: p < 0.05 vs. 10⁷ CFU/well 12-well.

Figure 8

Results show that 96-well recovery and lower E. coli density significantly increase the days to final percent positive rates. Days when individual Acanthamoeba ATCC 30461 trophozoite wells were identified as positive for each recovery method, within (A) Inoculum Control and (B) Solution 2 treatment. The percentage of positive wells was calculated using the total number of wells positive by Day 65 as the denominator and the positive wells positive on a particular day as the numerator. a: p < 0.05 vs. day 7 within the same recovery and E. coli density, b: p < 0.05 vs. Day 14 within the same recovery and E. coli density, c: p < 0.05 vs. 10⁷ CFU/well 96-well, d: p < 0.05 vs. 10⁷ CFU/well 12-well.

Figure 9

The density of Acanthamoeba in positive wells was visualized at the first day a representative well was identified as positive at a designated time point. Inoculum Control wells were evaluated for both strains (ATCC 30461 and ATCC 50370) and captured at 4× magnification. For ATCC 30461, all wells were captured at the Day 7 time point. For 50370, the 12-well and the 10⁷ CFU E. coli per well in the 96-well were captured at Day 7, while the two 10⁶ CFU E. coli in the 96-well were captured at Day 14. The density of Acanthamoeba in positive wells was visualized at the first day a representative well was identified as positive at a designated time point. Solution 2 wells were evaluated for both strains (ATCC 30461 and ATCC 50370) and captured at 4× magnification. All wells were captured at the Day 7 time point. Red arrows indicate only Acanthamoeba present, no other Acanthamoeba in the field of view; black arrows indicate a representative Acanthamoeba in the field. Green arrows indicate E.coli. Scale bar = 100 µm, all pictures taken at the same magnification. Reference Figure 6, Figure 7 and Figure 8 for quantification and analysis.

Figure 10

Representative images from ATCC 30461 and ATCC 50370 inoculum controls following seeding with a single Acanthamoeba trophozoite on Day 0. Inoculum Control wells from the 96-well containing Mid-10⁶ E. coli CFU were evaluated for both strains (ATCC 30461 and ATCC 50370) and captured at 4× magnification. ATCC 50370 was positive on Day 28 as indicated by the red arrows, which show all trophozoites in the field. ATCC 30461 was positive on day 7 with black arrows in Day 7 image indicating trophozoites only. On Day 14 and Day 21, the ATCC 30461 well had fully encysted, and the images show identical cysts at both time points. The single black arrows indicate the same cysts at both time points. Scale bar = 100 µm, all pictures taken at the same magnification. Green arrows indicate E.coli. See Figure 6, Figure 7 and Figure 8 for quantification and analysis.

Figure 11

Representative images from ATCC 50370: higher density E. coli results in earlier well positivity. (A,B) Inoculum Control wells from the 96-well containing Mid-10⁶ E. coli CFU from ATCC 50370 were captured at 4× magnification. (A) was positive at Day 35 as indicated by the arrows, while (B) was positive at Day 14. (C,D) Solution 2 wells from the 96-well containing Low 10⁶ E. coli CFU from ATCC 50370 were captured at 4× magnification. (C) was positive at Day 21, while (D) was positive at Day 14. Red arrows indicate all trophozoites present. Scale bar = 100 µm, all pictures taken at the same magnification. Green arrows indicate E.coli. See Figure 6, Figure 7 and Figure 8 for quantification and analysis.

Figure 12

Representative images of a Solution 2 well from a 96-well plate containing Low 10⁶ E. coli CFU from ATCC 50370 were captured at 4× magnification. The well was positive at Day 14 as indicated by the red arrows, which indicate all trophozoites present. Scale bar = 100 µm, all pictures taken at the same magnification. Green arrows indicate E.coli. See Figure 6, Figure 7 and Figure 8 for quantification and analysis.

Figure 13

Three potential life cycle paths for Acanthamoeba trophozoites, including the new path of dormancy (indicated by the dotted arrow), where Acanthamoeba trophozoites display extend proliferation suppression in the presence of a food source.

Figure 14

Experimental design system indicating recovery methods and plate counting. Each solution was tested with three independent replicates.

Figure 15

Single-cell experimental design with E. coli concentrations.