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Restriction enzyme analysis of mitochondrial DNA of Acanthamoeba strains in Japan
Abstract:
Eight isolates, identified as either Acanthamoeba castellanii or A. polyphaga from human eye infections, contact lens containers, and soil in Japan, were characterized by restriction fragment length polymorphisms (RFLP) of mitochondrial DNA (mtDNA). Mitochondrial DNA was digested with either Bgl II, EcoR I, Hind III, Hpa I, Sca I or Xba I, electrophoresed in agarose gels, and stained with ethidium bromide. Four distinct RFLP phenotypes that refer to the collection of six fragment size patterns obtained for a single strain with six enzymes, were discovered among the eight strains used in this study. Three strains morphologically classified as A. polyphaga share a single RFLP phenotype with the Ma strain of A. castellanii. The interspecific sequence differences of 7.06-12.74% in DNA nucleotide were estimated from the proportion of DNA fragments shared by each pair of mtDNA.
Insights
This study analyzed Acanthamoeba isolates from Japan using mitochondrial DNA restriction fragment length polymorphisms (RFLP). Four distinct RFLP patterns were found, revealing genetic diversity within these medically important amoebas.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Acanthamoeba species are opportunistic pathogens causing serious eye infections.
- Accurate identification of Acanthamoeba strains is crucial for understanding disease transmission and developing effective treatments.
- Previous classification relied heavily on morphology, which can be unreliable.
Purpose of the Study:
- To genetically characterize Acanthamoeba isolates from Japan using molecular methods.
- To differentiate between Acanthamoeba castellanii and Acanthamoeba polyphaga based on mitochondrial DNA (mtDNA) polymorphisms.
- To assess the genetic diversity within these clinically relevant amoeba populations.
Main Methods:
- Eight Acanthamoeba isolates from human eye infections, contact lens cases, and soil were analyzed.
- Mitochondrial DNA (mtDNA) was extracted and digested with six restriction enzymes (Bgl II, EcoR I, Hind III, Hpa I, Sca I, Xba I).
- Restriction fragment length polymorphisms (RFLP) were analyzed using agarose gel electrophoresis and ethidium bromide staining.
Main Results:
- Four distinct RFLP phenotypes were identified among the eight Acanthamoeba strains.
- Three strains identified morphologically as A. polyphaga shared an RFLP phenotype with the A. castellanii 'Ma' strain.
- Estimated interspecific sequence differences ranged from 7.06% to 12.74% based on shared DNA fragments.
Conclusions:
- Mitochondrial DNA RFLP is a valuable tool for differentiating Acanthamoeba species and strains.
- Genetic analysis revealed potential overlap or misclassification between A. polyphaga and A. castellanii based on morphology alone.
- This study highlights the genetic diversity of Acanthamoeba in Japan and its implications for clinical microbiology.