Because of an increased number of Acanthamoeba keratitis (AK) along with associated disease burdens, medical professionals have become more aware of this pathogen in recent years. In this study, by analyzing both the nuclear 18S small subunit ribosomal RNA (18S rRNA) and mitochondrial 16S rRNA gene loci, 27 clinical Acanthamoeba strains that caused AK in Japan were classified into 3 genotypes, T3 (3 strains), T4 (23 strains), and T5 (one strain). Most haplotypes were identical to the reference haplotypes reported from all over the world, and thus no specificity of the haplotype distribution in Japan was found. The T4 sub-genotype analysis using the 16S rRNA gene locus also revealed a clear sub-conformation within the T4 cluster, and lead to the recognition of a new sub-genotype T4i, in addition to the previously reported sub-genotypes T4a-T4h. Furthermore, 9 out of 23 strains in the T4 genotype were identified to a specific haplotype (AF479533), which seems to be a causal haplotype of AK. While heterozygous nuclear haplotypes were observed from 2 strains, the mitochondrial haplotypes were homozygous as T4 genotype in the both strains, and suggested a possibility of nuclear hybridization (mating reproduction) between different strains in Acanthamoeba. The nuclear 18S rRNA gene and mitochondrial 16S rRNA gene loci of Acanthamoeba spp. possess different unique characteristics usable for the genotyping analyses, and those specific features could contribute to the establishment of molecular taxonomy for the species complex of Acanthamoeba.
Acanthamoeba/classification/genetics/growth & development/*isolation & purification ; Acanthamoeba Keratitis/*parasitology ; Cell Nucleus/*genetics ; DNA, Mitochondrial/*genetics ; DNA, Protozoan/genetics ; Humans ; Japan ; Molecular Sequence Data ; Phylogeny ; RNA, Ribosomal, 16S/*genetics ; RNA, Ribosomal, 18S/*genetics

In a previous study, we reported our discovery of Acanthamoeba contamination in domestic tap water; in that study, we determined that some Acanthamoeba strains harbor endosymbiotic bacteria, via our molecular characterization by mitochondrial DNA restriction fragment length polymorphism (Mt DNA RFLP). Five (29.4%) among 17 Acanthamoeba isolates contained endosymbionts in their cytoplasm, as demonstrated via orcein staining. In order to estimate their pathogenicity, we conducted a genetic characterization of the endosymbionts in Acanthamoeba isolated from domestic tap water via 16S rDNA sequencing. The endosymbionts of Acanthamoeba sp. KA/WP3 and KA/WP4 evidenced the highest level of similarity, at 97% of the recently published 16S rDNA sequence of the bacterium, Candidatus Amoebophilus asiaticus. The endosymbionts of Acanthamoeba sp. KA/WP8 and KA/WP12 shared a 97% sequence similarity with each other, and were also highly similar to Candidatus Odyssella thessalonicensis, a member of the alpha-proteobacteria. The endosymbiont of Acanthamoeba sp. KA/WP9 exhibits a high degree of similarity (85-95%) with genus Methylophilus, which is not yet known to harbor any endosymbionts. This is the first report, to the best of our knowledge, to show that Methylophilus spp. can live in the cytoplasm of Acanthamoeba.
Acanthamoeba/isolation & purification/*microbiology/ultrastructure ; Alphaproteobacteria/classification/genetics/*isolation & purification ; Animals ; Bacteroidetes/classification/genetics/*isolation & purification ; Cluster Analysis ; DNA, Bacterial/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Fresh Water/*parasitology ; Korea ; Methylophilus/classification/genetics/*isolation & purification ; Microscopy, Electron, Transmission ; Phylogeny ; RNA, Ribosomal, 16S/genetics ; Sequence Analysis, DNA ; *Symbiosis

After morphological grouping of Acanthamoeba by Pussard and Pons, phylogeny of the genus has been always a big topic to the researchers. Because of the variability of morphological characteristics, unchangeable and stable characters have been investigated for phylogenic criteria. Isoenzyme and mitochondrial DNA RFLP (Mt DNA RFLP) analyses revealed different patterns among strains assigned to a same species. Therefore, these characteristics would be considered as tools for strain discrimination than species identification. The most recently developed and the most promising method is the sequence analysis of 18s ribosomal RNA coding DNA (18s rDNA). The phylogenic tree based on comparison of 18s rDNA sequences distinguished the 3 morphological groups of Acanthamoeba and divided them into 12 unique sequence types (T1-T12 genotypes). Most clinical and environmental isolates belonged to the morphological group II and the genotype T4. In the Republic of Korea, 2 strains of Acanthamoeba, YM-2 and YM-3, were first isolated from the environment in 1974. However, phylogenic identification of Korean Acanthamoeba isolates from human infections or the environment were tried from the late 1990s. By RFLP analysis or total sequence analysis of 18s rDNA revealed that almost all clinical isolates including the one from a suspicious granulomatous amebic encephalitis patient belonged to the genotype T4. A large number of environmental isolates from contact lens storage cases, tapped water, and ocean sediments also belonged to the genotype T4. Almost identical strain characteristics, such as Mt DNA RFLP pattern of environmental isolates, with the clinical isolates could make a simple conclusion that most environmental isolates might be a potential keratopathogen.
Acanthamoeba/*classification/genetics/*isolation & purification ; Amebiasis/parasitology ; DNA, Protozoan/genetics ; DNA, Ribosomal/genetics ; Humans ; Molecular Sequence Data ; *Phylogeny ; Soil/parasitology ; Water/parasitology

Three Acanthamoeba isolates (KA/E9, KA/E17, and KA/E23) from patients with keratitis were identified as Acanthamoeba triangularis by analysis of their molecular characteristics, a species not previously recognized to be a corneal pathogen. Epidemiologic significance of A. triangularis as a keratopathogen in Korea has been discussed. Morphologic features of Acanthamoeba cysts were examined under a microscope with differential interference contrast (DIC) optics. Mitochondrial DNA (mtDNA) of the ocular isolates KA/E9, KA/E17, and KA/E23 were digested with restriction enzymes, and the restriction patterns were compared with those of reference strains. Complete nuclear 18S and mitochondrial (mt) 16S rDNA sequences were subjected to phylogenetic analysis and species identification. mtDNA RFLP of 3 isolates showed very similar patterns to those of SH621, the type strain of A. triangularis. 16S and 18S rDNA sequence analysis confirmed 3 isolates to be A. triangularis. 18S rDNA sequence differences of the isolates were 1.3% to 1.6% and those of 16S rDNA, 0.4% to 0.9% from A. triangularis SH621. To the best of our knowledge, this is the first report, confirmed by 18S and 16S rDNA sequence analysis, of keratitis caused by A. triangularis of which the type strain was isolated from human feces. Six isolates of A. triangularis had been reported from contaminated contact lens cases in southeastern Korea.
Acanthamoeba/classification/genetics/isolation & purification ; Acanthamoeba Keratitis/*drug therapy/*parasitology ; Adolescent ; Adult ; Animals ; Antiprotozoal Agents/therapeutic use ; Biguanides/therapeutic use ; DNA, Mitochondrial/genetics ; DNA, Protozoan/genetics ; Female ; Humans ; Male ; Phylogeny ; Polymorphism, Restriction Fragment Length ; RNA, Ribosomal, 16S/genetics ; RNA, Ribosomal, 18S/genetics

The pathogenic mechanism of granulomatous amebic encephalitis (GAE) and amebic keratitis (AK) by Acanthamoeba has yet to be clarified. Protease has been recognized to play an important role in the pathogenesis of GAE and AK. In the present study, we have compared specific activity and cytopathic effects (CPE) of purified 33 kDa serine proteinases from Acanthamoeba strains with different degree of virulence (A. healyi OC-3A, A. lugdunensis KA/E2, and A. castellanii Neff). Trophozoites of the 3 strains revealed different degrees of CPE on human corneal epithelial (HCE) cells. The effect was remarkably reduced by adding phenylmethylsulfonylfluoride (PMSF), a serine proteinase inhibitor. This result indicated that PMSF-susceptible proteinase is the main component causing cytopathy to HCE cells by Acanthamoeba. The purified 33 kDa serine proteinase showed strong activity toward HCE cells and extracellular matrix proteins. The purified proteinase from OC-3A, the most virulent strain, demonstrated the highest enzyme activity compared to KA/E2, an ocular isolate, and Neff, a soil isolate. Polyclonal antibodies against the purified 33 kDa serine proteinase inhibit almost completely the proteolytic activity of culture supernatant of Acanthamoeba. In line with these results, the 33 kDa serine proteinase is suggested to play an important role in pathogenesis and to be the main component of virulence factor of Acanthamoeba.
Virulence Factors/isolation & purification/*metabolism ; Virulence ; Trophozoites/physiology ; Substrate Specificity ; Soil/parasitology ; Serine Endopeptidases/isolation & purification/*metabolism ; Humans ; Epithelial Cells/parasitology/*pathology ; Encephalitis ; Cornea/cytology/parasitology/*pathology ; Cells, Cultured ; Animals ; Acanthamoeba castellanii/enzymology/growth & development/pathogenicity ; Acanthamoeba Keratitis/parasitology ; Acanthamoeba/classification/*enzymology/growth & development/*pathogenicity

In an effort to characterize, on the molecular scale, the Acanthamoeba initially isolated from the cornea of an amoebic keratitis patient associated with overnight-wear orthokeratology lens in Korea, we conducted mitochondrial DNA restriction fragment length polymorphism, 18S rDNA sequencing, and drug sensitivity analyses on the isolate (KA/PE1). The patient was treated with polyhexamethylene biguanide, chlorhexidine and oral itraconazole, which resulted in resolution of the patient's ocular inflammation. The majority of the molecular characteristics of the KA/PE1 were determined to be identical, or quite similar, to those of A. castellanii Ma strain, which had been isolated also from amoebic keratitis. The risk of Acanthamoeba keratitis as a potential complication of overnight orthokeratology is briefly discussed.
*Sequence Analysis, DNA ; RNA, Ribosomal, 18S/genetics ; Polymorphism, Restriction Fragment Length ; Parasitic Sensitivity Tests ; Myopia/therapy ; Itraconazole/administration & dosage ; Humans ; Female ; Disinfectants/administration & dosage ; DNA, Ribosomal/analysis ; DNA, Protozoan/analysis ; DNA, Mitochondrial/analysis ; Contact Lenses/*adverse effects ; Chlorhexidine/administration & dosage ; Biguanides/administration & dosage ; Astigmatism/therapy ; Antiprotozoal Agents/administration & dosage ; Animals ; Adolescent ; Acanthamoeba Keratitis/drug therapy/*parasitology ; Acanthamoeba/classification/*genetics/*isolation & purification

The taxonomy of Acanthamoeba spp., an amphizoic amoeba which causes granulomatous amoebic encephalitis and chronic amoebic keratitis, has been revised many times. The taxonomic validity of some species has yet to be assessed. In this paper, we analyzed the morphological characteristics, nuclear 18s rDNA and mitochondrial 16s rDNA sequences and the Mt DNA RFLP of the type strains of four Acanthamoeba species, which had been previously designated as A. divionensis, A. parasidionensis, A. mauritaniensis, and A. rhysodes. The four isolates revealed characteristic group II morphology. They exhibited 18S rDNA sequence differences of 0.2-1.1% with each other, but more than 2% difference from the other compared reference strains. Four isolates formed a different clade from that of A. castellanii Castellani and the other strains in morphological group II on the phylogenetic tree. In light of these results, A. paradivionensis, A. divionensis, and A. mauritaniensis should be regarded as synonyms for A. rhysodes.
Acanthamoeba/*classification/*genetics ; Animals ; DNA, Mitochondrial/genetics ; DNA, Ribosomal/genetics ; Phylogeny ; Polymorphism, Restriction Fragment Length ; RNA, Ribosomal, 18S/genetics

Acanthamoeba and Naegleria are widely distributed in fresh water, soil and dust throughout the world, and cause meningoencephalitis or keratoconjunctivitis in humans and other mammals. Korean isolates, namely, Naegleria sp. YM-1 and Acanthamoeba sp. YM-2, YM-3, YM-4, YM-5, YM-6 and YM-7, were collected from sewage, water puddles, a storage reservoir, the gills of a fresh water fish, and by corneal washing. These isolates were categorized into three groups based on the mortalities of infected mice namely, highly virulent (YM-4), moderately virulent (YM-2, YM-5 and YM-7) and nonpathogenic (YM-3). In addition, a new species of Acanthamoeba was isolated from a freshwater fish in Korea and tentatively named Korean isolate YM-4. The morphologic characters of its cysts were similar to those of A. culbertsoni and A. royreba, which were previously designated as Acanthamoeba group III. Based on experimentally infected mouse mortality, Acanthamoeba YM-4 was highly virulent. The isoenzymes profile of Acanthamoeba YM-4 was similar to that of A. royreba. Moreover, an anti-Acanthamoeba YM-4 monoclonal antibody reacted only with Acanthamoeba YM-4, and not with A. culbertsoni. Random amplified polymorphic DNA marker analysis and RFLP analysis of mitochondrial DNA and of a 18S small subunit ribosomal RNA, placed Acanthamoeba YM-4 in a separate cluster based on phylogenic distances. Thus Acanthamoeba YM-4 was identified as a new species, and assigned Acanthamoeba sohi. Up to the year 2002 in Korea, two clinical cases were found to be infected with Acanthamoeba spp. These patients died of meningoencephalitis. In addition, one case of Acanthamoeba pneumonia with an immunodeficient status was reported and Acanthamoeba was detected in several cases of chronic relapsing corneal ulcer, chronic conjunctivitis, and keratitis.
*Acanthamoeba/classification/genetics/immunology/pathogenicity ; Amebiasis/diagnosis/epidemiology/*parasitology/therapy ; Animals ; Antigens, Protozoan/analysis/genetics/immunology ; DNA, Mitochondrial/analysis ; DNA, Protozoan/analysis ; Korea/epidemiology ; Life Cycle Stages ; *Naegleria/classification/genetics/immunology/pathogenicity ; Phylogeny ; Polymorphism, Restriction Fragment Length ; Random Amplified Polymorphic DNA Technique/veterinary ; Virulence/genetics

A new species of Acanthamoeba was isolated from a freshwater fish in Korea and tentatively named Acanthamoeba sp. YM-4 (Korean isolate YM-4). The trophozoites were 11.0-23.0 micrometer in length and had hyaline filamentous projections. Cysts were similar to those of A. culbertsoni and A. royreba, which were previously designated as Acanthamoeba group III. Acanthamoeba YM-4 can survive at 40 degrees C, and its generation time was 19.6 hr, which was longer than that of A. culbertsoni. In terms of the in vitro cytotoxicity of lysates, Acanthamoeba YM-4 was weaker than A. culbertsoni, but stronger than A. polyphaga. On the basis of the mortality of experimentally infected mice, Acanthamoeba YM-4 was found to be highly virulent. The isoenzymes profile of Acanthamoeba YM-4 was similar to that of A. royreba. An anti-Acanthamoeba YM-4 monoclonal antibody, McAY7, was found to react only with Acanthamoeba YM-4, and not with A. culbertsoni. Random amplified polymorphic DNA marker analysis and RFLP analysis of mitochondrial DNA and of 18S small subunit ribosomal RNA, placed Acanthamoeba YM-4 in a separate cluster on the basis of phylogenetic distances. Thus the Acanthamoeba Korean isolate YM-4 was identified as a new species, and assigned as Acanthamoeba sohi.
Acanthamoeba/*classification/genetics/isolation & purification/*pathogenicity ; Amebiasis/parasitology/*veterinary ; Animals ; DNA, Mitochondrial/analysis ; DNA, Protozoan/analysis ; Fish Diseases/*parasitology ; Gills/parasitology ; Goldfish/*parasitology ; Korea ; Mice ; Phylogeny ; Polymorphism, Restriction Fragment Length ; RNA, Ribosomal, 18S/genetics ; Random Amplified Polymorphic DNA Technique ; Virulence

We describe a riboprinting scheme for identification of unknown Acanthamoeba isolates at the species level. It involves use of the PCR-RFLP of small subunit ribosomal RNA gene (riboprint) of 24 reference strains by 4 kinds of restriction enzymes. Seven strains in morphological group I and III were identified at species level with their unique sizes of PCR product and riboprint type by Rsa I. Unique restriction fragment length patterns of 17 strains in group II by Dde I, Taq I and Hae III were classified into: (1) four taxa that were identifiable to the species level, (2) a subgroup of 4 taxa and a pair of 2 taxa that were identical to each other, and (3) a species complex of 7 taxa assigned to A. castellanii complex that were closely related. These results were consistent with that of 18s rDNA sequence analysis. This approach provides an alternative to the rDNA sequencing for rapid identification of a new clinical isolate or large number of environmental isolates of Acanthamoeba.
Acanthamoeba/classification/genetics/*isolation & purification ; Animals ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; RNA, Protozoan ; RNA, Ribosomal ; Ribotyping/*methods