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Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 2002 Apr;40(4):1199–1206. doi: 10.1128/JCM.40.4.1199-1206.2002

Mitochondrial DNA Restriction Fragment Length Polymorphism (RFLP) and 18S Small-Subunit Ribosomal DNA PCR-RFLP Analyses of Acanthamoeba Isolated from Contact Lens Storage Cases of Residents in Southwestern Korea

Hyun-Hee Kong 1, Ji-Yeol Shin 1, Hak-Sun Yu 1, Jin Kim 2, Tae-Won Hahn 3, Young-Ho Hahn 4, Dong-Il Chung 1,*
PMCID: PMC140361  PMID: 11923331

Abstract

We applied ribosomal DNA PCR-restriction fragment length polymorphism (RFLP) and mitochondrial DNA (mtDNA) RFLP analyses to 43 Acanthamoeba environmental isolates (KA/LH1 to KA/LH43) from contact lens storage cases in southwestern Korea. These isolates were compared to American Type Culture Collection strains and clinical isolates (KA/E1 to KA/E12) from patients with keratitis. Seven riboprint patterns were seen. To identify the species of the isolates, a phylogenetic tree was constructed based on the comparison of riboprint patterns with reference strains. Four types accounted for 39 of the isolates belonging to the A. castellanii complex. The most predominant (48.8%) type was A. castellanii KA/LH2 type, which had identical riboprint and mtDNA RFLP patterns to those of A. castellanii Castellani, KA/E3 and KA/E8. The riboprint and mtDNA RFLP patterns of the KA/LH7 (20.9%) type were identical to those of A. castellanii Ma, a corneal isolate from the United States. The riboprint and mtDNA RFLP patterns of the KA/LH1 (18.6%) type were the same as those of A. lugdunensis L3a, KA/E2, and KA/E12. The prevalent pattern for each type of Acanthamoeba in southwestern Korea was very different from that from southeastern Korea and Seoul, Korea. It is noteworthy that 38 (88.4%) out of 43 isolates from contact lens storage cases of the residents in southwestern Korea revealed mtDNA RFLP and riboprint patterns identical to those found for clinical isolates in our area. This indicates that most isolates from contact lens storage cases in the surveyed area are potential keratopathogens. More attention should be paid to the disinfection of contact lens storage cases to prevent possible amoebic keratitis.

Acanthamoeba spp., causative agents of the sight-threatening amoebic keratitis and the life-threatening granulomatous amoebic encephalitis, have ubiquitous distribution in human environments (35, 45). Amoebic keratitis was a rare corneal infection for 10 years after the first case report by Jones et al. (23). However, the number of reported cases has increased, apparently as a consequence of an association of the disease with contact lens wearing (36, 57). Amoebic keratitis has been continuously reported in Korea as well (unpublished data). Ecological studies (22, 30) showed that the contamination rate of contact lens paraphernalia by Acanthamoeba in Korea was much higher than rates reported in industrialized countries (24, 29, 56). In particular, the rate in southwestern Korea was twice that found in other provinces in Korea. The pathogenic potential of Acanthamoeba isolates from lens storage cases in Korea has yet to be determined. Because no simple and reliable in vivo or in vitro test exists, investigators have been using strain typing methods in order to indirectly assess the potential pathogenicity of the isolates (19, 49, 60).

Although the identification of Acanthamoeba at the genus level can be easily accomplished on the basis of the distinctive morphology of the trophozoites and cysts, there have been disputes over methods of species identification for Acanthamoeba. Pussard and Pons (42) classified Acanthamoeba into three groups according to the cyst size and morphological features. Group I consists of Acanthamoeba spp. with relatively large cysts, distinctly stellate endocysts, and smooth spherical ectocysts. Group II and group III Acanthamoeba spp. have smaller cysts (diameters less than 18 μm). Group II species have polygonal to stellate endocysts with irregular or wrinkled ectocysts, while the cysts of group III species have rounded or slightly angular endocysts with thinner and smooth or slightly wrinkled ectocysts. However, because of variable cyst morphology by culture conditions (53), species identification by morphology alone can hardly be possible (54).

Therefore, investigators have used several kinds of nonmorphological methods for the taxonomy of Acanthamoeba spp. Studies of isoenzyme patterns (11, 12, 14, 15, 55) suggested the other groupings of Acanthamoeba strains that are not consistent with previous species assignments based on morphological criteria. Kong et al. (28) and Chung et al. (6) reported intraspecific heterogeneity of the zymograms for several kinds of isoenzymes. Costas and Griffiths (13) considered that the variation among some Acanthamoeba species overlaps so extensively that the amoebae should be regarded as species complexes. Mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) analysis has also been applied for taxonomy analysis of Acanthamoeba (2, 3, 58, 59). However, considering the profound interstrain diversity of the mtDNA RFLP and alloenzyme profiles, Kong et al. (28) and Chung et al. (6) suggested that both analyses should be used for the strain identification, differentiation, and characterization rather than species identification.

The sequence of the small-subunit (ssu) rRNA gene is very useful as molecular data for phylogeny and taxonomy (5, 21, 33, 52). Stothard et al. (52) studied 18S (ssu) rRNA gene phylogeny of Acanthamoeba and classified 53 isolates into 12 sequence types. However, when the number of isolates to be studied is large, sequencing of the 2,300-bp rRNA gene is too labor-intensive, time-consuming, and expensive for identification or characterization of Acanthamoeba isolates. Thus, a simpler and less expensive method is needed. Riboprinting, the examination of restriction enzyme site polymorphism of ssu rRNA-coding DNA (rDNA) amplified by PCR, is a simple, inexpensive, and timely method and has recently been used to establish the taxonomic relationships among Entamoeba spp. (9, 10), among Naegleria spp. (8, 16), and among Acanthamoeba spp. (5, 27). Chung et al. (5) were the first to apply riboprinting to the subgenus classification of Acanthamoeba. Their results coincided well with those of rRNA sequencing performed by Stothard et al. (52). Acanthamoeba taxonomists generally agreed with the classification that Chung et al. (5) had suggested (T. J. Byers, personal communication). By either rDNA sequencing or riboprinting, most clinical isolates of morphological group II were shown to belong to a clade (T4 by Stothard et al., A. castellanii subgroup by Chung et al.) containing A. castellanii Castellani. Stothard et al. (52) suggested that various species in T4 all might be reclassified as A. castellanii because the sequence type includes the type strain for that species.

In the present study, we analyzed 43 Acanthamoeba isolates from contact lens storage cases of the residents of southwestern Korea using riboprinting and mtDNA RFLP analyses and evaluated the potential keratopathogenicities of these isolates.

MATERIALS AND METHODS

Acanthamoeba isolation and axenic culture.

In order to isolate Acanthamoeba from contaminated contact lens storage cases, the cases were opened under aseptic conditions. The centrifuged sediment from the solution in a case was aseptically smeared on a nonnutrient agar plate covered with heat-treated Escherichia coli (60). A sterile cotton ball was then rubbed over the internal surface of the lens case and placed on a different agar plate covered with heat-treated E. coli. The plates were incubated at 25°C for 1 week and examined daily for the presence and growth of Acanthamoeba under an inverted microscope.

The Acanthamoeba cysts encysted on the plate were grouped by their size and morphological features according to the criteria of Pussard and Pons (42).

A piece of agar plate (0.5 by 1 cm) covered with the cysts of a clone was treated with 0.1 N HCl for 24 h for axenization and washed with glass-distilled water three times. The piece of agar plate with many cysts was placed in Proteose Peptone-yeast extract-glucose medium (60) at 25°C.

Reference strains.

Twenty-three Acanthamoeba strains (Table 1) were obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). Twelve clinical isolates (KA/E1 to KA/E12) from Korean keratitis patients were also used as reference strains.

TABLE 1.

List of Acanthamoeba reference strains obtained from the ATCC

Strain ATCC no. Virulence Environmental source Geographic source Former species designation Reference no.
Castellani 30011 + Yeast culture England A. castellanii 17
L3a 50240 + Swimming pool France A. lugdunensis 42
Vil3 50241 NDa Swimming pool France A. quina 42
Jones 30461 + Keratitis United States A. polyphaga 23
SH621 50254 ND Human feces France A. triangularis 42
Nagington 30873 + Keratitis England A. polyphaga 38
Singh 30973 − Soil England A. rhysodes 50
1652 50253 − Soil England A. mauritaniensis 42
AA2 50238 − Soil Morocco A. divionensis 42
AA1 50251 − Soil France A. paradivionensis 42
Neff 30010 − Soil France A. castellanii 39
Ma 50370 + Keratitis United States A. castellanii 34
P23 30871 − Freshwater United States A. polyphaga 41
Chang 30898 + Freshwater United States A. castellanii 4
BH-2 30730 + Ocean sediment United States A. hatchetti 48
RB-F-1 50388 ND Ocean sediment United States A. stevensoni 47
S-7 30731 + Beach bottom United States A. griffini 46
Ray & Hayes 30137 ND Soil United States A. astronyxis 43
OC-15C 30867 ND River United States A. tubiashi 32
A-1 30171 + Tissue culture United States A. culbertsoni 51
OC-3A 30866 + GAEb United States A. healyi 37
GE-3a 50252 − Swimming pool France A. pustulosa 42
Reich 30870 − Soil Israel A. palestinensis 44
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a

aND, not determined.

b

bGAE, granulomatous amoebic encephalitis.

Riboprinting: PCR-RFLP of the ssu rDNA.

Chromosomal DNA of each clone was extracted as described by Kong and Chung (27). Amoebae harvested at the end of the logarithmic growth phase were washed with cold phosphate-buffered saline three times and boiled in 0.1 ml of 0.1 N NaOH for 3 min. The supernatant was collected after centrifugation at 800 × g for 2 min at room temperature and mixed with 0.2 ml of glass-distilled water. An equal volume of phenol was added to the solution, and this mixture was vortexed for 1 min. The mixture was centrifuged at 15,000 rpm for 5 min at 4°C twice. The resulting aqueous phase was centrifuged at 12,000 rpm for 5 min again with 300 μl of phenol-chloroform (1:1) solution. The nuclear DNA was precipitated by adding 600 μl of cold absolute ethanol and 30 μl of 3 M sodium acetate (pH 5.2), incubating this mixture at −70°C for 15 min, and then centrifuging it at 15,000 rpm for 20 min at 4°C. The sediment was washed with 70% ethanol, vacuum dried, and then dissolved in 30 to 50 μl of glass-distilled water. The DNA was stored at −20°C until used.

The sequence of primers for PCR ssu rDNA (P3, 5′-CCGAATTCGTCGACAACTGTTGATCCTGCCAGT-3′; P4, 5′-GGATCCAAGCTTGATCCTTC TGCAGGTTCACCTAC-3′) are designed to hybridize at the highly conserved sequences of the extreme 5′ (P3) and 3′ (P4) termini of eukaryotic ssu rDNA (1, 5). PCR consisted of 1 min at 94°C, 1 min at 58°C, and 2 min at 72°C in a thermal cycler (PE480; Perkin-Elmer Cetus). After 30 cycles, 10 min of extension time was given. After amplification, the PCR products of 43 Acanthamoeba isolates were checked by electrophoresis in a 1.5% agarose gel at 4 V/cm for 1.5 h. The amplified ssu rDNA was examined by digestion with 10 restriction endonucleases (MspI, HaeIII, HhaI, HinfI, DdeI, TaqI, Tru9I, MboI, RsaI, and Sau96I; Poscochem, Seongnam, Korea) that have recognition sequences of four nucleotides for 2 h. The digested DNA was electrophoresed in a 2.5% agarose gel (3 parts agarose, 1 part Nusieve). When the enzyme digested the DNA into small and similarly sized fragments, 15% polyacrylamide gel with Tris-borate-EDTA buffer was used to obtain clearer and more-accurate data of fragments. The ethidium bromide-stained gel was examined and photographed under a UV transilluminator.

Sequence divergence estimates were calculated by the Nei and Li equation (40) from the fragment comigration data set which was obtained by comparison of the riboprints of 43 isolates and all reference strains (5). A phylogenetic tree was constructed by the unweighted pair group method with arithmetic average using a computer program (PHYLIP, version 3.5) (7, 18).

mtDNA extraction and RFLP.

mtDNA of Acanthamoeba isolates was extracted by the method described by Yagita and Endo (59). Briefly, Acanthamoeba trophozoites washed with phosphate-buffered saline (pH 7.4) were suspended in 100 μl of chilled TEG buffer (25 mM Tris-HCl, 10 mM EDTA, 50 mM glucose; pH 8.0) and incubated on ice for 5 min. Amoebae were lysed by adding 200 μl of chilled fresh 1% sodium dodecyl sulfate solution in 0.2 N NaOH and then incubated subsequently on ice for 5 min. Chilled 3 M potassium acetate buffer was mixed with the suspension and incubated on ice for 15 min. Degraded cellular proteins and genomic DNA were extracted with equal volumes of phenol-chloroform by centrifugation at 12,000 rpm at 4°C for 5 min. mtDNA in supernatant was precipitated by adding 1.0 ml of absolute ethanol and 40 μl of 3 M sodium acetate solution and by incubating at −70°C for 15 min. After centrifugation at 15,000 rpm for 20 min at 4°C, the precipitated DNA was washed with 70% chilled ethanol. The DNA sediment was vacuum dried and dissolved in 15 to 25 μl of TE buffer (5 mM Tris-HCl, pH 8.0; 1 mM EDTA) and stored at −20°C until used. mtDNAs of 43 Acanthamoeba isolates were digested with restriction enzymes that have recognition sequences of six nucleotides at 37°C for 2 h (sometimes overnight) in a 20-μl reaction volume with the buffers specified for each restriction enzyme (EcoRI, BglII, XbaI, SalI, ScaI, ClaI, HpaI, and PvuII; Poscochem). Digested DNA was electrophoresed in a 0.7% agarose gel and stained with ethidium bromide. The RFLP of mtDNA of 43 strains was observed and photographed under a UV transilluminator. The HindIII-digested λ phage was used as a size marker.

RESULTS

Morphology of Acanthamoeba isolates.

Among 43 isolates analyzed in this study, 42 isolates belonged to the morphological group II of Pussard and Pons (42). In their system, the cyst has a polygonal endocyst and an irregularly wrinkled thick ectocyst, with the cyst diameter ranging from 11.8 to 16.5 μm and the number of arms ranging from 3.8 to 6.2. One other isolate (KA/LH35) belonged to group III, which features round endocysts closely attached to wrinkled ectocysts.

Analysis of ssu rDNA PCR-RFLP.

Figure 1 presents PCR-amplified full-length rDNA and RFLP patterns obtained by using three kinds of restriction enzymes. The size of the PCR products was approximately 2,300 bp in all isolates, meaning no intron was present in the ssu rDNAs of 43 environmental isolates. Considering all patterns by eight kinds of restriction enzymes (some data not shown), a total of seven different rDNA RFLP types emerged. Using the phylogenetic tree of the isolates with reference strains based on the estimated sequence divergence, 41 isolates were identified as A. castellanii complex, and 1 was identified as A. polyphaga (Fig. 2), but the remaining isolate has yet to be studied. Among the 41 isolates belonging to A. castellanii complex, 8 isolates (KA/LH1 type) showed the same rDNA PCR-RFLP pattern as A. lugdunensis L3a. Twenty-one isolates (KA/LH2 type) showed patterns of rDNA PCR-RFLP very similar to those of A. castellanii Castellani, the type strain of the type species. Nine isolates (KA/LH7 type) were identical to A. castellanii Ma in their rDNA PCR-RFLP pattern. The pattern of one isolate (KA/LH32 type) was identical to that of A. castellanii Castellani. An isolate (KA/LH35 type) with morphological characteristics of group III showed a unique rDNA PCR-RFLP pattern. Two isolates (KA/LH39 type), although they belonged to A. castellanii complex, showed a rDNA PCR-RFLP pattern somewhat different from that of the Castellani strain. One isolate (KA/LH8 type) clustered with the A. rhysodes clade.

FIG. 1.

FIG. 1.

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Agarose gel electrophoretic pattern of PCR products from seven different types of Acanthamoeba and their polyacrylamide gel electrophoretic restriction fragment patterns. Lanes M, HindIII-digested λ phage DNA or 100-bp ladder (DNA size standards).

FIG. 2.

FIG. 2.

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Phylogenetic tree of seven different types and 23 reference strains of Acanthamoeba based on the rDNA PCR-RFLP analyses obtained by using the unweighted pair group method with arithmetic average and a computer program (PHYLIP, version 3.5).

mtDNA restriction phenotypes.

Figure 3 shows agarose gel electrophoretic patterns of EcoRI digests of mtDNA from 43 Acanthamoeba isolates. They were divided into seven different types according to their patterns. Although KA/LH2 and KA/LH7 types appeared to have almost the same patterns by EcoRI digestion, as seen in Fig. 3, the fragment sizes were slightly different. Moreover, they showed very different RFLP patterns with other restriction enzymes, such as BglII (Fig. 4A). The most predominant type was KA/LH2, and 21 isolates showed the same mtDNA RFLP pattern. Nine isolates were of the KA/LH7 type, and eight isolates were of the KA/LH1 type. Two isolates, KA/LH39 and KA/LH40, had the same mtDNA RFLP pattern as each other. The mtDNA RFLP pattern of KA/LH32 was very similar to that of KA/LH2 but slightly different. The other two isolates, KA/LH8 and KA/LH35, had unique mtDNA RFLP patterns. A total of seven different restriction phenotypes by BglII enzymes emerged for 43 isolates, as shown in Fig. 4A. The other three kinds of restriction enzymes we tested showed the same patterns (data not shown).

FIG. 3.

FIG. 3.

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Agarose gel electrophoretic restriction fragment patterns by EcoRI of mtDNA of 43 environmental Acanthamoeba isolates. Lanes M, HindIII-digested λ phage DNA (DNA size standard).

FIG. 4.

FIG. 4.

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Agarose gel electrophoretic restriction fragment patterns of mtDNA by BglII restriction enzyme. Seven different types of 43 environmental Acanthamoeba isolates (A), three types of environmental isolates and associated reference strains of Acanthamoeba (B), and two types of environmental isolates and associated clinical isolates of Acanthamoeba from keratitis patients in Korea (C) were compared. Lanes: M, HindIII-digested λ phage DNA (DNA size standard); L3a, A. lugdunensis L3a; Castellani, A. castellanii Castellani; Ma, A. castellanii Ma.

DISCUSSION

In the present study, 43 Acanthamoeba isolates, originating from contact lens storage cases of residents in southwestern Korea, were morphologically and genetically analyzed based on riboprinting and mtDNA RFLP analysis. Except for one isolate (KA/LH35), all belonged to the morphological group II (42). These isolates may be morphologically assigned to the A. castellanii or A. polyphaga complexes. This indicates that Acanthamoeba of morphological group II may be predominant in contact lens storage cases in Korea. Based on riboprints, 41 isolates were genetically very closely related to A. castellanii Castellani, and 1 was closely related to A. polyphaga. The remaining 1 isolate showed a unique riboprint. This coincided well with the results of morphological observation and indicated that amoebae closely related to A. castellanii may be predominant in contact lens storage cases in Korea.

Although rDNA sequencing or riboprinting is promising for subgenus classification of Acanthamoeba (5, 19, 52), it is not simple to identify isolates at the species level, especially strains in group II, to which most clinical and environmental isolates belong. Stothard et al. (52) suggested that various species in T4 might be reclassified as A. castellanii because the sequence type includes the type strain for that species. However, the molecular characteristics of strains in T4 were quite different among strains, and many species which belonged to this clade have already been assigned. We would rather assign this subgroup (T4 sequence type) as a species complex (60), as Costas and Griffiths suggested (13).

Based on the riboprints and mtDNA RFLP analysis in the present study, the A. castellanii KA/LH2 type was the most predominant (48.8%) type of Acanthamoeba from contact lens storage cases in southwestern Korea (Table 2). It is noteworthy that the riboprint and mtDNA RFLP patterns of the KA/LH2 type were identical to those of A. castellanii Castellani, KA/E3, and KA/E8 (Fig. 4B and C)—the type strain and two clinical isolates from infected corneas of Korean patients, respectively. The predominance of the Castellani type of Acanthamoeba in southwestern Korea is unique compared to the previous survey results in southeastern and Seoul, Korea. The predominance of the Castellani type was 3.6% in the southeastern area (31) and 7.7% in Seoul (60).

TABLE 2.

Isolates of Acanthamoeba spp. from contact lens storage cases and RFLP phenotypes of their rDNA and mt-DNA

Isolate rDNA RFLP phenotype mtDNA RFLP phenotype Finally assigned species Associated pathogenic strain(s)
KA/LH1 KA/LH1 KA/LH 1 A. castellanii complex KA/E2, KA/E12
KA/LH2 KA/LH2 KA/LH 2 A. castellanii complex KA/E3, KA/E8
KA/LH3 KA/LH2 KA/LH 2 A. castellanii complex KA/E3, KA/E8
KA/LH4 KA/LH2 KA/LH 2 A. castellanii complex KA/E3, KA/E8
KA/LH5 KA/LH2 KA/LH 2 A. castellanii complex KA/E3, KA/E8
KA/LH6 KA/LH2 KA/LH 2 A. castellanii complex KA/E3, KA/E8
KA/LH7 KA/LH7 KA/LH 7 A. castellanii complex Ma
KA/LH8 KA/LH8 KA/LH 8
KA/LH9 KA/LH7 KA/LH 7 A. castellanii complex Ma
KA/LH10 KA/LH2 KA/LH 2 A. castellanii complex KA/E3, KA/E8
KA/LH11 KA/LH1 KA/LH 1 A. castellanii complex KA/E2, KA/E12
KA/LH12 KA/LH2 KA/LH 2 A. castellanii complex KA/E3, KA/E8
KA/LH13 KA/LH7 KA/LH 7 A. castellanii complex Ma
KA/LH14 KA/LH1 KA/LH 1 A. castellanii complex KA/E2, KA/E12
KA/LH15 KA/LH7 KA/LH 7 A. castellanii complex Ma
KA/LH16 KA/LH2 KA/LH 2 A. castellanii complex KA/E3, KA/E8
KA/LH17 KA/LH 2 KA/LH 2 A. castellanii complex KA/E3, KA/E8
KA/LH18 KA/LH 2 KA/LH 2 A. castellanii complex KA/E3, KA/E8
KA/LH19 KA/LH 1 KA/LH 1 A. castellanii complex KA/E2, KA/E12
KA/LH20 KA/LH 7 KA/LH 7 A. castellanii complex Ma
KA/LH21 KA/LH 2 KA/LH 2 A. castellanii complex KA/E3, KA/E8
KA/LH22 KA/LH 2 KA/LH 2 A. castellanii complex KA/E3, KA/E8
KA/LH23 KA/LH 2 KA/LH 2 A. castellanii complex KA/E3, KA/E8
KA/LH24 KA/LH 2 KA/LH 2 A. castellanii complex KA/E3, KA/E8
KA/LH25 KA/LH 7 KA/LH 7 A. castellanii complex Ma
KA/LH26 KA/LH 2 KA/LH 2 A. castellanii complex KA/E3, KA/E8
KA/LH27 KA/LH 1 KA/LH 1 A. castellanii complex KA/E2, KA/E12
KA/LH28 KA/LH 2 KA/LH 2 A. castellanii complex KA/E3, KA/E8
KA/LH29 KA/LH 1 KA/LH 1 A. castellanii complex KA/E2, KA/E12
KA/LH30 KA/LH 7 KA/LH 7 A. castellanii complex Ma
KA/LH31 KA/LH 2 KA/LH 2 A. castellanii complex KA/E3, KA/E8
KA/LH32 KA/LH 32 KA/LH 32 A. castellanii
KA/LH33 KA/LH 2 KA/LH 2 A. castellanii complex KA/E3, KA/E8
KA/LH34 KA/LH 2 KA/LH 2 A. castellanii complex KA/E3, KA/E8
KA/LH35 KA/LH 35 KA/LH 35
KA/LH36 KA/LH 7 KA/LH 7 A. castellanii complex Ma
KA/LH37 KA/LH 2 KA/LH 2 A. castellanii complex KA/E3, KA/E8
KA/LH38 KA/LH 1 KA/LH 1 A. castellanii complex KA/E2, KA/E12
KA/LH39 KA/LH 39 KA/LH 39 A. castellanii complex
KA/LH40 KA/LH 39 KA/LH 39 A. castellanii complex
KA/LH41 KA/LH 2 KA/LH 2 A. castellanii complex KA/E3, KA/E8
KA/LH42 KA/LH 7 KA/LH 7 A. castellanii complex Ma
KA/LH43 KA/LH 1 KA/LH 1 A. castellanii complex KA/E2, KA/E12
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Acanthamoeba castellanii KA/LH7 (20.9%) and KA/LH1 (18.6%) types were the second and third most prevalent types of Acanthamoeba in the surveyed area. The riboprint and mtDNA RFLP patterns of the KA/LH7 type were identical to those of A. castellanii Ma (Fig. 4B), a clinical isolate from the United States (34). The Ma type has never been isolated in southeastern Korea (31) but was relatively prevalent (20.5%) in Seoul (60). The riboprint and mtDNA RFLP patterns of the KA/LH1 type were the same as those of A. castellanii L3a, KA/E2, and KA/E12 (Fig. 4B and C). The KA/LH1 type was reported to be the most prevalent type of Acanthamoeba in southeastern Korea (64.3%) and in Seoul (51.3%). Two amoebic keratitis patients from Seoul and Pusan were recently reported to be infected with this type (unpublished data).

The prevalent pattern for each type of Acanthamoeba in southwestern Korea was very different from those in the southeastern area. However, it was interesting that the pattern in Seoul was intermediate between those in the southeastern and southwestern areas, because the citizens in Seoul are composed of people from various provinces, including provinces in the southeastern and southwestern areas. The prevalence pattern can vary from nation to nation. Kilvington et al. (25) reported that isolates with the mtDNA RFLP type of A. castellanii Jones (formerly A. polyphaga or A. lugdunensis) were demonstrated the most frequently among clinical isolates of Acanthamoeba in Europe. According to Gautom et al. (20), isolates with the mtDNA restriction phenotype of the A. castellanii Castellani type were demonstrated the most frequently from clinical and environmental sources in the United States. In Japan, isolates with mtDNA RFLP patterns of the A. castellanii Ma type were demonstrated the most frequently from contact lens paraphernalia and infected corneas (T. Endo, personal communication).

The riboprint pattern of the KA/LH32 type was the same as that of the Castellani, KA/E3, and KA/E8 types, but the KA/LH32 type could not be regarded as a clone of the Castellani, KA/E3, or KA/E8 types but was regarded as a closely related clone, because the mtDNA RFLP pattern of the KA/LH32 type was slightly different from those of the reference strains. This finding can be explained by the fact that mtDNA evolves faster than nuclear DNA. Therefore, the mitochondrial genome can be used for phylogenetic study of closely related Acanthamoeba isolates. The mtDNA RFLP pattern of KA/LH32 was almost identical to that of JAC/E1, an isolate from a Japanese keratitis patient (58).

The most important fact in the present study is that 38 (88.4%) out of 43 isolates from contact lens storage cases of the residents of southwestern Korea revealed mtDNA RFLP and riboprint patterns identical to those of clinical isolates of Acanthamoeba. This indicates that most isolates in this study are potential keratopathogens. Four of the remaining five isolates were new types.

In contrast to the high contamination rate of contact lens paraphernalia by potentially keratopathogenic Acanthamoeba spp. in Korea, the incidence of Acanthamoeba keratitis so far is low. It is suggested that the combination of wearing a contact lens contaminated with keratopathogenic Acanthamoeba and minor injury of the corneal epithelium may be the predisposing factor in Acanthamoeba keratitis among contact lens wearers. At any rate, more attention should be paid to the disinfection of contact lens storage cases to prevent possible amoebic keratitis.

Acknowledgments

This work was supported by a grant (HMP-97-2-0029 for the 1997, Good Health R&D Project) from the Ministry of Health and Welfare, Republic of Korea.

REFERENCES

  • 1.Bhattacharya, D., T. Helmchen, and M. Melkonian. 1998. Molecular evolutionary analyses of nuclear-encoded small subunit ribosomal RNA identify and independent rhizopod lineage containing the Euglyphina and the Chlorarachniophyta. J. Eukaryot. Microbiol. 42:65-69. [DOI] [PubMed] [Google Scholar]
  • 2.Bogler, S. A., C. D. Zarley, L. L. Burianek, P. A. Fuerst, and T. J. Byers. 1983. Interstrain mitochondrial DNA polymorphism detected in Acanthamoeba by restriction endonuclease analysis. Mol. Biochem. Parasitol. 8:145-163. [DOI] [PubMed] [Google Scholar]
  • 3.Byers, T. J., S. A. Bogler, and L. L. Burianek. 1983. Analysis of mitochondrial DNA variation as an approach to systematic relationships in the genus Acanthamoeba. J. Protozool. 30:198-203. [Google Scholar]
  • 4.Byers, T. J., E. R. Hugo, and V. J. Stewart. 1990. Genes of Acanthamoeba: DNA, RNA and protein sequence. J. Protozool. 37:s17-s25. [DOI] [PubMed] [Google Scholar]
  • 5.Chung, D. I., H. S. Yu, M. Y. Hwang, T. H. Kim, T. O. Kim, H. C. Yun, and H. H. Kong. 1998. Subgenus classification of Acanthamoeba by riboprinting. Korean J. Parasitol. 36:69-80. [DOI] [PubMed] [Google Scholar]
  • 6.Chung, D. I., H. H. Kong, H. S. Yu, Y. M. Oh, S. T. Yee, and Y. J. Lim. 1996. Biochemical and molecular characterization of a strain KA/S2 of Acanthamoeba castellanii isolated from Korean soil. Korean J. Parasitol. 34:79-85. [DOI] [PubMed] [Google Scholar]
  • 7.Clark, C. G. 1992. Riboprinting: a molecular approach to the identification and taxonomy of protozoa, p. D-4.1-D-4.4. In J. J. Lee and A. T. Soldo (ed.), Protocols in protozoology, vol. 1. Allen Press, Lawrence, Kans.
  • 8.Clark, C. G., G. A. M. Cross, and J. R. De Jonckheere. 1989. Evaluation of evolutionary divergence in the genus Naegleria by analysis of ribosomal DNA plasmid restriction patterns. Mol. Biochem. Parasitol. 34:281-296. [DOI] [PubMed] [Google Scholar]
  • 9.Clark, C. G., and L. S. Diamond. 1991. The Laredo strain and other ‘Entamoeba histolytica-like’ amoebae are Entamoeba moskovskii. Mol. Biochem. Parasitol. 46:11-18. [DOI] [PubMed] [Google Scholar]
  • 10.Clark, C. G., and L. S. Diamond. 1991. Ribosomal RNA genes of ‘pathogenic’ and ‘nonpathogenic’ Entamoeba histolytica are distinct. Mol. Biochem. Parasitol. 49:297-303. [DOI] [PubMed] [Google Scholar]
  • 11.Costas, M., and A. J. Griffiths. 1980. The suitability of starch gel electrophoresis of esterases and acid phosphatases for the study of Acanthamoeba taxonomy. Arch. Protistenkd. 123:272-279. [Google Scholar]
  • 12.Costas, M., and A. J. Griffiths. 1985. Enzyme composition and the taxonomy of Acanthamoeba. J. Protozool. 18:399-405. [DOI] [PubMed] [Google Scholar]
  • 13.Costas, M., and A. J. Griffiths. 1986. Physiological characterization of Acanthamoeba strains. J. Protozool. 33:304-309. [Google Scholar]
  • 14.Culbertson, C. G., P. W. Ensminger, and W. M. Overton. 1965. The isolation of additional strains of pathogenic Hartmanella sp. (Acanthamoeba). Am. J. Clin. Pathol. 35:383-387. [DOI] [PubMed] [Google Scholar]
  • 15.De Jonckheere, J. F. 1983. Isoenzyme and total protein analysis by agarose isoelectric focusing, and taxonomy of the genus Acanthamoeba. J. Protozool. 30:701-706. [Google Scholar]
  • 16.De Jonckheere, J. F. 1994. Riboprinting of Naegleria spp.: small-subunit versus large-subunit rDNA. Parasitol. Res. 80:230-234. [DOI] [PubMed] [Google Scholar]
  • 17.Douglas, M. 1930. Notes on the classification of the amoeba found by Castellani in cultures of a yeast-like fungus. J. Trop. Med. London 33:258-259. [Google Scholar]
  • 18.Felsenstein, J. 1993. PHYLIP manual, version 3.5. Department of Genetics, University of Washington, Seattle.
  • 19.Gast, R. J., D. LeDee, P. A. Fuerst, and T. J. Byers. 1996. Subgenus systematics of Acanthamoeba: four nuclear 18S rDNA sequence types. J. Eukaryot. Microbiol. 43:498-504. [DOI] [PubMed] [Google Scholar]
  • 20.Gautom, R. K., S. Lory, S. Seyedirashti, D. L. Bergeron, and T. R. Fritsche. 1994. Mitochondrial DNA fingerprinting of Acanthamoeba spp. isolated from clinical and environmental sources. J. Clin. Microbiol. 32:1070-1073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Grimont, F., and P. A. D. Grimont. 1986. Ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools. Ann. Microbiol. (Inst. Pasteur) 137:165-175. [DOI] [PubMed] [Google Scholar]
  • 22.Hahn, T. W., D. I. Chung, H. H. Kong, and Y. H. Han. 1998. Nation-wide survey for Acanthamoeba from contact lens case systems in Korea. J. Korean Ophthalmol. 39:667-672. [Google Scholar]
  • 23.Jones, D. B., G. S. Visvesvara, and N. M. Robinson. 1975. Acanthamoeba polyphaga keratitis and Acanthamoeba uveitis associated with fatal meningo-encephalitis. Trans. Ophthalmol. Soc. United Kingdom 95:221-232. [PubMed] [Google Scholar]
  • 24.Kato, T., H. Shiota, Y. Mimura, Y. Ito, S. Tei, M. Sakamoto, and T. Miwatani. 1990. Microbial contamination of stock solution for soft contact lens. Jpn. J. Clin. Ophthalmol. 44:1037-1040. [Google Scholar]
  • 25.Kilvington, S., J. R. Beeching, and D. G. White. 1991. Differentiation of Acanthamoeba strains from infected corneas and the environment by using restriction endonuclease digestion of whole-cell DNA. J. Clin. Microbiol. 29:310-314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Kingston, D., and D. C. Warhurst. 1969. Isolation of amoebae from the air. J. Med. Microbiol. 2:27-36. [DOI] [PubMed] [Google Scholar]
  • 27.Kong, H. H., and D. I. Chung. 1996. PCR and RFLP variation of conserved region of small subunit ribosomal DNA among Acanthamoeba isolates assigned to either A. castellanii or A. polyphaga. Korean J. Parasitol. 34:127-134. [DOI] [PubMed] [Google Scholar]
  • 28.Kong, H. H., J. H. Park, and D. I. Chung. 1995. Interstrain polymorphisms of isoenzyme profiles and mitochondrial DNA fingerprints among seven strains assigned to Acanthamoeba polyphaga. Korean J. Parasitol. 33:331-340. [DOI] [PubMed] [Google Scholar]
  • 29.Larkin, D. F. P., S. Kilvington, and D. L. Easty. 1990. Contamination of contact lens storage cases by Acanthamoeba and bacteria. Br. J. Ophthalmol. 74:133-135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Lee, S. M., Y. J. Choi, and D. I. Chung. 1996. Contamination of contact lens care system with special reference to Acanthamoeba. Korean J. Ophthalmol. 38:756-761. [Google Scholar]
  • 31.Lee, S. M., Y. J. Choi, H. W. Ryu, H. H. Kong, and D. I. Chung. 1997. Species identification and molecular characterization of Acanthamoeba isolated from contact lens paraphernalia. Korean J. Ophthalmol. 11:39-50. [DOI] [PubMed] [Google Scholar]
  • 32.Lewis, E. J., and T. K. Sawyer. 1979. Acanthamoeba tubiashi n. sp. a new species of fresh-water Amoebida (Acanthamoebidae). Trans. Am. Microsc. Soc. 98:543-549. [Google Scholar]
  • 33.Liesack, W., and E. Stackebrandt. 1992. Occurrence of novel groups of the domain Bacteria as revealed by analysis of genetic material isolated from an Australian terrestrial environment. J. Bacteriol. 174:5072-5078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Ma, P., E. Willaert, K. B. Juechter, and A. R. Stevens. 1981. A case of keratitis due to Acanthamoeba in New York, and features of 10 cases. J. Infect. Dis. 143:662-667. [DOI] [PubMed] [Google Scholar]
  • 35.Martinez, A. J., and G. S. Visvesvara. 1997. Free-living, amphizoic and opportunistic amebas. Brain Pathol. 7:583-598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Moore, M. B., J. P. McCulley, C. Newton, L. M. Cobo, G. N. Foulks, D. M. O'Day, K. J. Johns, W. T. Driebe, L. A. Wilson, R. J. Epstein, and D. J. Doughman. 1987. Acanthamoeba keratitis: a growing problem in soft and hard contact lens wearers. Ophthalmology 94:1654-1661. [PubMed] [Google Scholar]
  • 37.Moura, H., S. Wallace, and G. S. Visvesvara. 1992. Acanthamoeba healyi n. sp. and the isoenzyme and immunoblot profiles of Acanthamoeba spp., groups 1 and 3. J. Protozool. 39:573-583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Nagington, J., P. G. Watson, T. J. Playfair, J. McGill, and B. R. Jones. 1974. Amebic infection of the eye. Lancet ii:1537-1540. [DOI] [PubMed] [Google Scholar]
  • 39.Neff, R. 1957. Purification, axenic cultivation and description of a soil amoeba. Acanthamoeba sp. J. Protozool. 4:176-182. [Google Scholar]
  • 40.Nei, M., and W. H. Li. 1979. Mathematical model for studying genetic variation in terms of restriction endonuclease. Proc. Natl. Acad. Sci. USA 76:5269-5273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Page, F. C. 1967. Re-definition of the genus Acanthamoeba with descriptions of three species. J. Protozool. 14:709-724. [DOI] [PubMed] [Google Scholar]
  • 42.Pussard, M., and R. Pons. 1977. Morphologie de la paroi kystique et taxonomie du genre Acanthamoeba (Protozoa, Amoebida). Protistologica 13:557-598. [Google Scholar]
  • 43.Ray, D. L., and R. E. Hayes. 1954. Hartmannella astronyxis: a new species of free-living amoeba. Cytology and life cycle. J. Morphol. 95:159-188. [Google Scholar]
  • 44.Reich, K. 1933. Studien er die Bodenprotozoen palstinos. Arch. Protistenkd. 79:76-98. [Google Scholar]
  • 45.Rosenberg, A. S., and M. B. Morgan. 2001. Disseminated acanthamoebiasis presenting as lobular panniculitis with necrotizing vasculitis in a patient with AIDS. J. Cutan. Pathol. 28:307-313. [DOI] [PubMed] [Google Scholar]
  • 46.Sawyer, T. K. 1971. Acanthamoeba griffini, a new species of marine amoeba. J. Protozool. 18:650-654. [Google Scholar]
  • 47.Sawyer, T. K., T. A. Nerad, E. J. Lewis, and S. M. McLaughlin. 1993. Acanthamoeba stevensoni n. sp. (Protozoa: Amoebida) from sewage-contaminated shellfish beds in Raritan Bay, New York. J. Protozool. 40:742-746. [Google Scholar]
  • 48.Sawyer, T. K., G. S. Visvesvara, and B. A. Harke. 1977. Pathogenic amoebas from brackish and ocean sediments, with a description of Acanthamoeba hatchetti, n. sp. Science 196:1324-1325. [DOI] [PubMed] [Google Scholar]
  • 49.Schroeder, J. M., G. C. Booton, J. Hay, I. A. Niszl, D. V. Seal, M. B. Markus, P. A. Fuerst, and T. J. Byers. 2001. Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of Acanthamoeba from humans with keratitis and from sewage sludge. J. Clin. Microbiol. 39:1903-1911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Singh, B. N. 1952. Nuclear division in nine species of small free-living amoebae and its bearing on the classification of the order Amoebida. Phil. Trans. R. Soc. London Ser. B 236:405-461. [DOI] [PubMed] [Google Scholar]
  • 51.Singh, B. N., and S. R. Das. 1970. Studies on pathogenic and nonpathogenic small free-living amoebae and the bearing of nuclear division on the classification of the order Amoebida. Phil. Trans. R. Soc. London Ser. B 259:435-476. [DOI] [PubMed] [Google Scholar]
  • 52.Stothard, D. R., J. M. Schroeder-Diedrich, M. H. Awwad, R. J. Gase, D. R. Ledee, S. Rodriguez-zaragoza, C. L. Dean, P. A. Fuerst, and T. J. Byers. 1998. The evolutionary history of the genus Acanthamoeba and the identification of eight new 18S rRNA gene sequence types. J. Eukaryot. Microbiol. 45:45-54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Stratford, M. P., and A. J. Griffiths. 1978. Variations in the properties and morphology of cysts of Acanthamoeba castellanii. J. Gen. Microbiol. 108:33-37. [Google Scholar]
  • 54.Visvesvara, G. S. 1991. Classification of Acanthamoeba. Rev. Infect. Dis. 13(Suppl. 5):S369-S372. [DOI] [PubMed] [Google Scholar]
  • 55.Visvesvara, G. S., S. S. Mirra, F. H. Brandt, D. M. Moss, H. M. Mathews, and A. J. Martines. 1983. Isolation of two strains of Acanthamoeba castellanii from human tissue and their pathogenicity and isoenzyme profiles. J. Clin. Microbiol. 18:1405-1412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Watanabe, R., Y. Ishibashi, S. Hommura, and K. Ishii. 1994. Acanthamoeba isolation from contact lens solution of contact lens wearers without keratitis. J. Jpn. Ophthalmol. Soc. 98:477-480. [PubMed] [Google Scholar]
  • 57.Wilhelmus, K. R. 1991. International symposium on Acanthamoeba and the eye. Introduction: the increasing importance of Acanthamoeba. Rev. Infect. Dis. 13(Suppl. 5):367-368. [DOI] [PubMed] [Google Scholar]
  • 58.Yagita, K. 1993. Characterization of Acanthamoeba isolates from eye infection and the environment by restriction endonuclease digestion of mitochondrial DNA. Jpn. J. Parasitol. 42:468-478. [Google Scholar]
  • 59.Yagita, K., and T. Endo. 1990. Restriction enzyme analysis of mitochondrial DNA of Acanthamoeba strains in Japan. J. Protozool. 37:570-575. [DOI] [PubMed] [Google Scholar]
  • 60.Yu, H. S., K. H. Choi, H. K. Kim, H. H. Kong, and D. I. Chung. 2001. Genetic analyses of Acanthamoeba isolates from contact lens storage cases of students in Seoul, Korea. Korean J. Parasitol. 39:161-170. [DOI] [PMC free article] [PubMed] [Google Scholar]

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