Genotypic diversity of Acanthamoeba strains isolated from Chilean patients with Acanthamoeba keratitis

Collection of clinical specimens

The samples received for Acanthamoeba diagnosis at the ISPCh were processed as described previously [4]. Briefly, the samples were inoculated onto 2% non-nutrient agar plates overlaid with 100 μl of a liquid culture of Escherichia coli in Page’s solution (NNA-E. coli). The plates were incubated at 37 °C for seven days and examined daily under a conventional light microscope for the presence of trophozoites and/or cyst. All positive culture plates were routinely collected in Page’s solution, subcultured in 5 ml Petri dishes with NNA overlaid with live E. coli, and also diluted 1:2 with sterile glycerol for long term storage at -20 °C. Amoebas from subculture plates were harvested and rinsed three times in phosphate-buffered saline (pH 7.4) and then transferred to Eppendorf tubes for further molecular analysis. When the genotyping service was not readily accessible, the samples were stored frozen until the service became available. At that time, an aliquot of the frozen stock was inoculated onto NNA overlaid with live E. coli until growth was observed. Then, the samples of amoeba were collected and processed for molecular analysis as described above.

DNA extraction and PCR amplification assay

Acanthamoeba trophozoites and cysts were lysed by thermal shock at 56 °C for 15 min with proteinase K; DNA was then extracted using a QIAamp DNA Mini Kit ® (Qiagen, Venlo, NLD) following the manufacturer’s protocol.

The ASA.S1 region in Rns was amplified using the Acanthamoeba genus-specific primers, forward JDP1 (5'-GGC CCA GAT CGT TTA CCG TGA A-3') and reverse JDP2 (5'-TCT CAC AAG CTG CTA GGG GAG TCA-3') as described by Booton et al. [10]. These primers amplified a fragment of approximately 500 bp. The PCR reactions were set up to a final volume of 50 μl, using 30 ng DNA, 1× amplification buffer [10 mM Tris-HCl (pH 9.0), 3.5 mM MgCl₂], 2 mM of each dNTP, 20 μM of each primer, 1.25 U Taq DNA polymerase and distilled water to make the volume. Amplification cycles were performed in a GeneAmp System 2700 thermocycler (Applied Biosystems, Foster City, CA, USA). The PCR cycles were set up as follows: pre-denaturation step at 94 °C for 3 min and 35 cycles of denaturation at 94 °C for 30 s, 60 °C of annealing for 30 s and then 72 °C for 1 min, followed by a final elongation step of 72 °C for 5 min. T4 Acanthamoeba strains previously identified at our laboratory were used as a positive control and DNA-free water was used as a negative control. The amplicons were resolved by agarose-gel electrophoresis and visualized using ethidium-bromide.

Sequencing and genotyping of strains

PCR products were purified using a DNA band purification kit (Omega Bio-Tek, Omega, Norcross, GA, USA), according to manufacturer’s instructions, and sequenced in both directions. The sequences of the PCR amplicons were obtained using the ABI PRISM BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) with 5 pmol of JDP1 and JDP2 primers and a 310 ABI PRISM Genetic Analyzer.

Alignment and data exploration

The sequences were assembled and edited with ALIGN, EditSeq and MegAlign (DNASTAR, Madison, WI, USA).

Phylogenetic analysis

The Bayesian inference algorithms implemented in MrBayes v.3.0B4 were used to infer phylogenetic trees [14]. A total of 131 related nucleotide sequences of the Rns gene were selected considering an E-value close to zero and percentage identity > 30% [15].

The multiple alignment of the nucleotide sequences was performed in BioEdit and corrected in GeneDoc v.2.7.000 [16]. After including gaps to maximize alignments, the final number of nucleotide positions was 459 bp. For the phylogenetic analysis, we used Acanthamoeba T14 (Acanthamoeba sp. AF333609 and AF333607) genotypes as outgroups, following to phylogenetic tree obtained by Risler et al. [17]. In addition, we included genotypes T5 (A. lenticulata U94741), T2 (Acanthamoeba sp. AB425949; Acanthamoeba polyphaga ATCC30872, AY026244), T10 (A. healyi AF019070; Acanthamoeba sp. GU808320), T11 (A. stevensoni AF019069; Acanthamoeba sp. GU808311), T13 (Acanthamoeba sp. AY102615 and AF132136) and T16 (Acanthamoeba sp. AY026245 and GQ380408). The best-fitting model of nucleotide substitution was selected using the Bayesian information criterion implemented in the program MEGA v.7 [18, 19]. These results gave the best fit for the K2+Γ nucleotide substitution model [20].

We simultaneously executed three parallel MCMC runs, each for 30 × 10⁶ generations with four Markov chains (one cold and three hot chains). Each run was analyzed in Tracer to confirm that effective sample sizes (ESS) were sufficient for all parameters (posterior ESS values > 300) [21]. The nodes were considered supported if posterior probabilities (PP) > 0.95. The trees were visualized using FigTree v.1.1 available at http://tree.bio.ed.ac.uk/software/figtree/ [21].

AK patients and samples

Genotyping of the AK isolates and sequence analysis

The molecular identification of the Acanthamoeba isolates was confirmed by the detection of the PCR fragment of the expected size for all isolates (423–551 bp). The sequences of the ASA.S1 amplicons obtained in this study have been deposited in the GenBank database (accession numbers are shown in Table 2). By sequencing analysis of the DF3 region, 108 samples from the 73 patients were identified as genotype T4. Five strains from two patients were identified as Acanthamoeba sp. T11, while a single isolate obtained from a patient was identified as T2 genotype (Table 2).

The subgenus classification of the T4 isolates was done by the sequence analysis of a 54–69 bp long variable region of the DF3, which revealed the presence of 24 sequence types, herein referred to as T4/A to X. The alignment of a highly variable section of the DF3 fragments showed four new sequence types (T4/L, S, T and U) that have not been previously described (Additional file 1: Table S1).

T4/A was the most prevalent sequence type being identified in 41 (38%) samples isolated from 28 (38.4%) patients. Furthermore, this T4/A variant was most frequent among the corneal scrape isolates (19 out of 55 samples, 35.6%). The sequence type T4/B was identified in isolates from 8 patients; the sequence types C and D were obtained from five patients each; sequence types E, F and G were isolated from 4, 3 and 6 patients, respectively; sequence types H, M and U were found in samples from 2 patients each; while the remaining sequence types were identified in isolates from individual patients.

Most of the samples obtained from the same patient had the same type of T4 variant; however, in five cases (patient IDs: 54, 57, 58, 72 and 76) the samples presented different variants.

Phylogenetic analysis

A total of 128 samples were included in this analysis (114 isolates obtained in this study and 14 reference strains; see methods). The phylogenetic tree constructed considering the partial sequence of the Rns gene (Fig. 1) was consistent with previous reports [17]. The tree showed well-supported clusters for each of the genotypes (PP ≥ 0.95). The sequences from patient samples used in this study were clustered according to genotypes T2 (n = 1), T11 (n = 5), and T4 (n = 108).

In relation to T4 genotype, the phylogenetic tree recovered three groups, two of them were well supported. The first group was weakly supported (PP = 0.52) and included the variants T4/C, D, I, L and Q. The second group (PP = 0.95) included variants T4H, J, O, P, R, T and U. Finally, the third group (PP = 0.95) included variants T4A, B, E, F, G, K, M, N, S, V, W and X.