Isolation and identification of Acanthamoeba strains from soil and tap water in Yanji, China.

Background Members of the genus Acanthamoeba are widely distributed throughout the world, and some of them are considered pathogenic, as they are capable of causing corneal and central nervous system diseases. In this study, we isolated Acanthamoeba strains from soil and tap water in Yanji, China. Methods We identified four strains of Acanthamoeba (CJY/S1, CJY/S2, CJY/S3, and CJY/W1) using mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) analysis. Nuclear 18S rDNA sequences were used for phylogenetic analysis and species identification. Results Genotypic characterization of the isolates showed that they belonged to genotypes T4 (CJY/S1 and CJY/S2), T5 (CJY/S3), and T16 (CJY/W1). Sequence differences between CJY/S1 and Acanthamoeba castellanii Neff, CJY/S2 and Acanthamoeba KA/E7, and CJY/S3 and Acanthamoeba lenticulata 68–2 were 0.31, 0.2, and 0.26%, respectively. 18S ribosomal deoxyribonucleic acid (rDNA) of CJY/W1 had 99% sequence identity to that of Acanthamoeba sp. U/H-C1. Strains CJY/S1 and CJY/S2, isolated from soil, had similar mtDNA RFLP patterns, whereas strain CJY/W1, isolated from tap water, displayed a different pattern. Conclusions To the best of our knowledge, this is the first report on the identification of genotypes T4, T5, and T16 from environmental sources in Yanji, China.

We identified these species by using mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) analysis and 18S rDNA sequence alignment. We found that the three strains isolated from the soil belonged to the morphological group II and had genotypes T4 and T5, whereas the strain isolated from tap water belonged to the morphological group II and had genotype T16.

Isolation and cultivation of Acanthamoeba
Samples of soil and tap water were collected in Yanji, China. The samples were loaded onto 1.5% agar plates covered with heat-inactivated (60°C for 1 h) Escherichia coli (American Type Culture Collection, ATCC 25922, free of plasmid). The plates were incubated at 25°C, and growth of Acanthamoeba was observed under an inverted microscope on a daily basis for 1 week. Each cyst isolated with a glass capillary was inoculated on a new agar plate and incubated for 1 week. For axenization, a piece of agar (1 cm × 1 cm) covered with cysts was treated with 0.1 N HCl for 24 h, washed three times with sterile water, placed in peptone yeast glucose medium (10 g proteose peptone, 10 g yeast extract, 10 mL of 50% glucose, 10 mL of 0.5 M Na 2 HPO 4 , and 10 mL of 0.5 M K 2 HPO 4 in 970 mL of sterile water), and incubated at 25°C for 3 weeks. When most of the amoebae reached trophozoite stage, they were harvested and washed three times with phosphate-buffered saline.

Morphological examination
A cyst formed on the monoxenic plate was picked with a sterilized inoculating loop and transferred to a glass slide with a drop of sterile distilled water. The slide was then covered with a coverslip. Fifty cysts per plate were observed and measured under a Nomarski (differential interference contrast) microscope (Olympus, Japan).

Analysis of 18S rDNA sequences
Genomic DNA was extracted using phenol/chloroform method. The 18S rRNA gene was amplified using the following primers by Xuan et al. [13]: forward, 5′-CC GAATTCGTCGACAACCTGGTTGATCCTGCCAGT-3′; reverse, 5′-GGATCCAAGCTTGATCCTTCTGCAGGTT CACCTAC-3′. The amplified products were resolved by electrophoresis, recovered from the gel, and ligated into a T/A cloning vector (pGEM-T Easy Vector System I, Promega, USA) for subsequent transformation of E. coli. Positive clones were picked, and recombinant plasmid DNA was extracted using the Wizard® Plus Minipreps DNA Purification System (Promega, USA). Plasmids with inserts of the correct size were identified by EcoRI digestion and sequenced. The obtained final 18S ribosomal DNA (rDNA) sequences of the Acanthamoeba strains were deposited in GenBank (accession nos. KY827389-KY827392). The sequences were then compared with those of other Acanthamoeba sequences in GenBank using the Basic Local Alignment Search Tool (BLAST) search engine. Clustal X and GeneDoc were used for pairwise alignment and calculation of percent sequence dissimilarity. Phylogenetic analyses were performed, and the phylogenetic tree was drawn using the neighbor-joining (NJ) method with MEGA3 [15]. Extraction of mtDNA and RFLP analysis mtDNA of Acanthamoeba isolates was extracted using the method described by Yagita and Endo [16]. Briefly, amoebae were harvested and washed in cold phosphatebuffered saline, treated with TEG buffer (25 mM Tris-HCl, 10 mM ethylenediaminetetraacetic acid (EDTA), 50 mM glucose, pH 8.0), lysed with a fresh solution of 1% sodium dodecyl sulfate in 0.2 N NaOH and potassium acetate buffer, and left on ice for 30 min. mtDNA was then extracted with a phenol/chloroform mixture (1:1) and recovered by the precipitation with cold absolute ethanol in the presence of sodium acetate. The extracted mtDNA was then digested with EcoRI at 37°C. The digested mtDNA was electrophoresed in 0.7% agarose gel and stained with ethidium bromide. The mtDNA RFLP patterns were observed and photographed.

Morphology of Acanthamoeba isolates
Cysts of the Acanthamoeba isolates CJY/S1, CJY/S2, CJY/S3, and CJY/W1 exhibited morphological characteristics typical of group II, as defined by Pussard and Pons [17]. They exhibited double-walled cyst morphology and featured thick, wrinkled ectocysts and satellite or polygonal endocysts (Fig. 1). The cyst diameter varied from 12.0 to 18.8 μm, and the number of arms was four to six (Table 1).

Discussion
There are many species of free-living amoebae. Some, such as Acanthamoeba spp., Naegleria spp., Hartmannella spp., or Balamuthia mandrillaris, are opportunists that can cause infections in humans and animals [18,19]. Naegleria and Acanthamoeba species have been identified as causes of serious human infections. Some species of Acanthamoeba can cause amoebic keratitis, particularly in contact lens wearers and immunocompromised individuals experiencing subacute or chronic central nervous system infections [20,21].
Eighteen species of Acanthamoeba have been classified into three groups according to the shape and size of cysts. Species in group I are nonpathogenic except A. astronyxis, A. byersi and A. comandoni [22,23]. Most of the pathogenic Acanthamoeba species belong to group II. Species in group III, such as A. culbertsoni, A. healyi, and A. lenticulata, often cause infections of the brain. The cyst morphology of the four isolates characterized in this study resembled that of various species within morphological group II. However, the classification of Acanthamoeba spp. based on morphological characteristics has proven to be unreliable. The morphology of Acanthamoeba spp. may change depending on culture conditions. Furthermore, different Acanthamoeba species in the same group can have similar morphology, and Acanthamoeba cysts of two species may show only transient differences, thereby causing difficulties in the identification of the species.
Lass et al. reported partial sequences of T4 strains in environmental samples in China recently [14], which is different from our data which is full of 18S sequences of four distinct genotypes of isolated strains. At present, sequence analysis of genomic DNA is considered the method of choice for identifying species of Acanthamoeba. Sequence analysis of 18S rRNA genes is frequently used.
Recent studies have shown that A. castellanii (genotype T4) and A. lenticulata (genotype T5) can infect the cornea and central nervous system in humans, but there are insufficient reports pertaining to strains of genotype T16. Further research is required to determine whether the Acanthamoeba sp. strains CJY/S1, CJY/S2, CJY/S3, and CJY/W1 isolated by us from environmental samples could be pathogenic to humans and animals.