Genetic analyses of Acanthamoeba isolates from contact lens storage cases of students in Seoul, Korea

Abstract

We conducted both the small subunit ribosomal DNA (SSU rDNA) polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and mitochondrial (mt) DNA RFLP analyses for a genetic characterization of Acanthamoeba isolates from contact lens storage cases of students in Seoul, Korea. Twenty-three strains of Acanthamoeba from the American Type Culture Collection and twelve clinical isolates from Korean patients were used as reference strains. Thirty-nine isolates from contact lens storage cases were classified into seven types (KA/LS1, KA/LS2, KA/LS4, KA/LS5, KA/LS7, KA/LS18, KA/LS31). Four types (KA/LS1, KA/LS2, KA/LS5, KA/LS18) including 33 isolates were regarded as A. castellanii complex by riboprints. KA/LS1 type was the most predominant (51.3%) in the present survey area, followed by KA/LS2 (20.9%), and KA/LS5 (7.7%) types. Amoebae of KA/LS1 type had the same mtDNA RFLP and riboprint patterns as KA/E2 and KA/E12 strains, clinical isolates from Korean keratitis patients. Amoebae of KA/LS2 type had the identical mtDNA RFLP patterns with A. castellanii Ma strain, a corneal isolate from an American patient as amoebae of KA/LS5 type, with KA/E3 and KA/E8 strains from other Korean keratitis patients. Amoebae of KA/LS18 type had identical patterns with JAC/E1, an ocular isolate from a Japanese patient. Three types, which remain unidentified at species level, were not corresponded with any clinical isolate in their mtDNA RFLP and riboprint patterns. Out of 39 isolates analyzed in this study, mtDNA RFLP and riboprint patterns of 33 isolates (84.6%) were identical to already known clinical isolates, and therefore, they may be regarded as potentially keratopathogenic. These results suggest that contact lens wearers in Seoul should pay more attention to hygienic maintenance of contact lens storage cases for the prevention of Acanthamoeba keratitis.

Fig. 1

Agarose gel electrophoretic restriction fragment patterns by EcoR I of mitochondrial DNA of Acanthamoeba 39 isolates. M, Hind III digested λ phage DNA as a DNA size standard.

Fig. 2

Agarose gel eletrophoretic restriction fragment patterns of mitochondrial DNA of seven different types of Acanthamoeba by eight kinds of restriction enzymes. M, Hind III digested λ phage DNA as a DNA size standard. Lane 1, KA/LS1; 2, KA/LS2; 3, KA/LS5; 4, KA/LS4; 5, KA/LS18; 6, KA/LS7; 7, KA/LS31.

Fig. 3

(A) Agarose gel electrophoretic pattern of PCR products of SSU rDNA of 7 types of Acanthamoeba isolates; M, Hind III digested λ phage DNA. (B)(C) Electrophoretic patterns of restriction fragments of PCR product from 7 types of Acanthamoeba isolates by Dde I and Hinf I; B, Agarose gel; C, Polyacrylamide gel; M, 50 bp ladder (BM, Germany); Lane 1, KA/LS1; 2, KA/LS2; 3, KA/LS5; 4, KA/LS4; 5, KA/LS18; 6. KA/LS7; 7, KA/LS31.

Fig. 4

UPGMA phenogram of 7 different types and 23 reference strains of Acanthamoeba based on the SSU rDNA PCR-RFLP analyses. ◎; 7 different types of Acanthamoeba isolates from contact lens storage cases.

Fig. 5

Comparative mitochondrial DNA RFLP patterns of Acanthamoeba isolated from contact lens storage cases with those of reference strains from keratitis. Lane 1, KA/LS1; 2, KA/E2; 3, KA/LS2; 4, Ma; 5, KA/LS5; 6, KA/E3; 7, KA/LS4; 8, KA/LS18; 9, KA/LS7; 10, KA/LS31; M, Hind III digested λ phage DNA.