-
The Korean Journal of Parasitology Sep 1999We investigated the value of mitochondrial small subunit rRNA gene (mt SSU rDNA) PCR-RFLP as a taxonomic tool for Acanthamoeba isolates with close inter-relationships....
We investigated the value of mitochondrial small subunit rRNA gene (mt SSU rDNA) PCR-RFLP as a taxonomic tool for Acanthamoeba isolates with close inter-relationships. Twenty-five isolates representing 20 species were included in the analysis. As in nuclear 18S rDNA analysis, two type strains (A. astronyxis and A. tubiashi) of morphological group 1 diverged earliest from the other strains, but the divergence between them was less than in 18S riboprinting. Acanthamoeba griffini of morphological group 2 branched between pathogenic (A. culbertsoni A-1 and A. healyi OC-3A) and nonpathogenic (A. palestinensis Reich, A. pustulosa GE-3a, A. royreba Oak Ridge, and A lenticulata PD2S) strains of morphological group 3. Among the remaining isolates of morphological group 2, the Chang strain had the identical mitochondrial riboprints as the type strain of A. hatchetti. AA2 and AA1, the type strains of A. divionensis and A. paradivionensis, respectively, had the identical riboprints as A. quina Vil3 and A. castellanii Ma. Although the branching orders of A. castellanii Neff, A. polyphaga P23, A. triangularis SH621, and A. lugdunensis L3a were different from those in 18S riboprinting analysis, the results obtained from this study generally coincided well with those from 18S riboprinting. Mitochondrial riboprinting may have an advantage over nuclear 18S rDNA riboprinting because the mt SSU rDNAs do not seem to have introns that are found in the 18S genes of Acanthamoeba and that distort phylogenetic analyses.
Topics: Acanthamoeba; Animals; DNA, Mitochondrial; DNA, Protozoan; DNA, Ribosomal; Phylogeny; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length
PubMed: 10507226
DOI: 10.3347/kjp.1999.37.3.181 -
The Korean Journal of Parasitology Jun 1999To determine the pathogenicity of Acanthamoeba spp. isolated in Korea and to develop a isoenzymatic maker, the mortality rate of infected mice, in vitro cytotoxicity...
To determine the pathogenicity of Acanthamoeba spp. isolated in Korea and to develop a isoenzymatic maker, the mortality rate of infected mice, in vitro cytotoxicity against target cells and isoenzyme band patterns were observed. Five isolates of Acanthamoeba spp. (YM-2, YM-3, YM-4, YM-5, and YM-7) were used in this study as well as three reference Acanthamoeba spp. (A. culbertsoni, A. hatchetti, and A. royreba). According to the mortality rate of infected mice, Korean isolates could be categorized into three groups high virulent (YM-4), low virulent (YM-2, YM-5, YM-7) and the nonpathogenic group (YM-3). In addition, the virulence of Acanthamoeba spp. was enhanced by brain passage in mice. In the cytotoxicity assay against chinese hamster ovary cells, especially, the cytotoxicity of brain-passaged amoebae was relatively higher than the long-term cultivated ones. The zymodeme patterns of glucose-6-phosphate dehydrogenase (G6PD), malate dehydrogenase (MDH), hexokinase (HK), glutamate oxaloacetate transaminase (GOT) and malic enzyme (ME) of Acanthamoeba spp. were different among each isolate, and also between long-term cultured amoebae and brain passaged ones. In spite of the polymorphic zymodemes, a slow band of G6PD and HK, and an intermediate band of MDH were only observed in pathogenic Acanthamoeba spp., which should be used as isoenzymatic makers.
Topics: Acanthamoeba; Amebiasis; Animals; Aspartate Aminotransferases; CHO Cells; Cricetinae; Glucosephosphate Dehydrogenase; Hexokinase; Isoenzymes; Korea; Malate Dehydrogenase; Mice; Mice, Inbred ICR; Virulence
PubMed: 10388266
DOI: 10.3347/kjp.1999.37.2.85 -
The Korean Journal of Parasitology Jun 1998Subgenus classification of Acanthamoeba remains uncertain. Twenty-three reference strains of Acanthamoeba including 18 (neo)type-strains were subjected for...
Subgenus classification of Acanthamoeba remains uncertain. Twenty-three reference strains of Acanthamoeba including 18 (neo)type-strains were subjected for classification at the subgenus level by riboprinting. PCR/RFLP analysis of 18S rRNA gene (rDNA). On the dendrogram reconstructed on the basis of riboprint analyses, two type-strains (A. astronyxis and A. tubiashi) of morphological group 1 diverged early from the other strains and were quite distinct from each other. Four type-strains of morphological group 3, A. culbertsoni, A. palestinensis, A. healyi were considered taxonomically valid, but A. pustulosa was regarded as an invalid synonym of A. palestinensis. Strains of morphological group 2 were classified into 6 subgroups. Among them, A. griffini which has an intron in its 18S rDNA was the most divergent from the remaining strains. Acanthamoeba castellanii Castellani, A. quina Vil3, A. lugdunensis L3a, A. polyphaga Jones, A. triangularis SH621, and A. castellanii Ma strains belonged to a subgroup, A. castellanii complex. However, A. quina and A. lugdunensis were regarded as synonyms of A. castellanii. The Chang strain could be regarded as A. hatchetti. Acanthamoeba mauritaniensis, A. divionensis, A. paradivionensis could be considered as synonyms of A. rhysodes. Neff strain was regarded as A. polyphaga rather than as A. castellanii. It is likely that riboprinting can be applied for rapid identification of Acanthamoeba isolated from the clinical specimens and environments.
Topics: Acanthamoeba; Animals; DNA, Protozoan; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; RNA, Protozoan; RNA, Ribosomal, 18S
PubMed: 9637824
DOI: 10.3347/kjp.1998.36.2.69 -
Korean Journal of Ophthalmology : KJO Jun 1997We applied ribosomal RNA gene (rDNA) polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to identify Acanthamoeba isolates from contact lens...
We applied ribosomal RNA gene (rDNA) polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to identify Acanthamoeba isolates from contact lens paraphernalia, and characterized these on the basis of mitochondrial DNA (mtDNA) RFLP and isoenzyme analysis. The 22 Acanthamoeba strains used as reference strains were obtained from the American Type Culture Collection. Twenty-eight isolates were classified into six ribogroups, as follows: Acanthamoeba ribogroup (AcRG) 1 consisted of 18 isolates; AcRG 2, of three, AcRG 3, of three; AcRG 4, of two; AcRG 5, of one, and AcRG 6, of one. AcRG 1, which was the most frequently isolated type, was identified as A. lugdunensis, and AcRG 2 as A. hatchetti. AcRG 4 was identified as A. triangularis, while AcRG 3 and AcRG 5 were closely related to A. triangularis. AcRG 6 was identified as A. castellanii. The mtDNA RFLP patterns and zymograms for five isoenzymes of the isolates belonging to a ribogroup were identical to one another. The mtDNA digestion phenotype and zymogram for acid phosphatase (AcP) of AcRG 1 were identical to those of A. lugdunensis L3a and KA/E2, the type strain and corneal isolates from a Korean keratitis patient, respectively. The mtDNA digestion phenotype and zymogram for AcP of AcRG 6 were identical to those of A. castellanii Castellani and KA/E3, the type strain and another corneal isolate found in Korea, respectively. The mtDNA RFLP and zymogram for AcP of AcRG 2 were very similar to those of A. hatchetti BH-2 and Chang, respectively the type strain and a pathogen. The mtDNA RFLP and zymogram for AcP of AcRG 4 were similar to those of A. triangularis SH621, the type strain. The mtDNA RFLP patterns of AcRG 3 and 5 were unique. These results showed that the riboprints, mtDNA RFLP and zymograms of 22 of 28 Acanthamoeba isolates were the same as or very similar to those of the clinical isolates, which can probably be regarded as keratopathogens. More attention should be paid to the prevention of contamination by Acanthamoeba and to the disinfection of contact lens paraphernalia.
Topics: Acanthamoeba; Acanthamoeba Keratitis; Animals; Contact Lenses; DNA, Mitochondrial; DNA, Protozoan; Electrophoresis, Agar Gel; Equipment Contamination; Humans; Isoelectric Focusing; Isoenzymes; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; RNA, Protozoan; RNA, Ribosomal
PubMed: 9283153
DOI: 10.3341/kjo.1997.11.1.39 -
Cornea Jan 1997Effective chemotherapy for Acanthamoeba keratitis has been hampered because of the marked resistance of the parasites to a variety of antimicrobial agents. In view of... (Comparative Study)
Comparative Study
Effective chemotherapy for Acanthamoeba keratitis has been hampered because of the marked resistance of the parasites to a variety of antimicrobial agents. In view of the fact that topical Brolene (propamidine isethionate) and neosporin are currently considered to be the medical treatment of choice in Europe, we sought to determine whether pentamidine may be equally effective, because the drug is more readily available to ophthalmologists in the United States. In this study, we compared the amoebicidal activity of the Brolene (commercial product), propamidine isethionate and pentamidine isethionate (Pentam) in vitro against three different species of Acanthamoeba, and the drugs' corresponding biocompatibility with rabbit corneal epithelial and endothelial cell cultures. The results indicated that there were significant species differences in drug sensitivity. Propamidine (> 1,000 micrograms/ml) was clearly less effective than pentamidine (> 125 micrograms/ml) against A. castellanii, although equivalent potency (> 250 micrograms/ml) was observed against A. polyphaga. On the other hand, propamidine (> 31.25 micrograms/ml) was slightly more effective than pentamidine (> 62.5 micrograms/ml) against A. hatchetti. Both drugs were also relatively nontoxic after short-term contact with cell cultures, even though the highest concentration of pentamidine caused low-grade injury to the superficial epithelium and reversible membrane damage to the endothelium. Steady-state levels of propamidine at effective amoebicidal concentrations, however, were much more toxic than pentamidine, which indicated that the drug has a much lower therapeutic index. Our data suggest that pentamidine may be an effective therapeutic option because of its potency and low toxicity.
Topics: Acanthamoeba; Amebicides; Animals; Benzamidines; Cell Culture Techniques; Cornea; Endothelium, Corneal; Epithelium; Pentamidine; Rabbits
PubMed: 8985640
DOI: No ID Found -
Current Eye Research Feb 1996We determined the relative minimal inhibitory and minimal amoebicidal concentrations of chlorhexidine digluconate and polyhexamethylene biguanide for four species of...
We determined the relative minimal inhibitory and minimal amoebicidal concentrations of chlorhexidine digluconate and polyhexamethylene biguanide for four species of Acanthamoeba. The amoebae were grown in peptone-glucose-yeast extract broth for 72 h in tissue culture flasks. Either washed trophozoites (approximately 10(5)) or cysts (approximately 10(5)) were incubated in the enrichment broth in 96 well microtiter trays. Antimicrobial concentrations of the biguanides were determined from microscopic examinations of methylene blue uptake and from subcultures. In general, killing was time dependent. Minimal amoebicidal concentrations at 24 h ranged from 50 to 100 mg/ml and to as low as 25 mg/ml by 72 h. Trophozoites were killed more rapidly than cysts. Both biguanides had similar levels of activity. A synergistic combination of chlorhexidine and polyhexamethylene biguanide (total concentration 25 mg/ml) was most evident for A. castellanii and A. polyphaga. Cysts of A. culbertsoni and A. hatchetti stained more rapidly after exposure to the combination of biguanides than to the single biguanides, but there were no statistically significant differences in the final numbers of dead or stained cysts after exposure to the combination or to the single biguanides.
Topics: Acanthamoeba; Amebicides; Animals; Biguanides; Chlorhexidine; Disinfectants; Dose-Response Relationship, Drug; Drug Synergism; Osmolar Concentration; Time Factors
PubMed: 8670733
DOI: 10.3109/02713689608997418 -
The Korean Journal of Parasitology Dec 1995Genetic status of Acanthamoeba spp. were tested on the basis of random amplified polymorphic DNA (RAPD) marker analysis. Four previously established Acanthamoeba...
Genetic status of Acanthamoeba spp. were tested on the basis of random amplified polymorphic DNA (RAPD) marker analysis. Four previously established Acanthamoeba species, 4 Korean isolates of Acanthamoeba sp., and one American isolate of Acanthamoeba sp. were analyzed by RAPD-PCR using an arbitrary decamer primers. Amplification products were fractionated by agarose gel electrophoresis and stained by ethidium bromide. Eighteen primers produced DNA amplification profiles revealing clear differences among 4 species. Nine of them also produced DNA amplification profiles which included some isolate-specific amplification products. On the basis of amplified fragments by 18 primers, the pairwise similarity indices between A. culbertsoni and other species (i.e., A. hatchetti, A. triangularis, A. polyphaga) were 0.300, 0.308, and 0.313, respectively. Similarity index between A. hatchetti and A. triangularis was 0.833. The mean similarity index among the 3 Korean isolates (YM-2, -3, -4) was 0.959 and 0.832 among them and 2 other species (A. hatchetti and A. triangularis). The mean similarity index among YM-5 and other Korean isolates (YM-2, -3, -4) was 0.237. However, the similarity index between YM-5 and A. culbertsoni was 0.857, which suggests that YM-5 is genetically more similar to A. culbertsoni than other Korean isolates. Phenogram reconstructed by UPGMA method revealed that there are two groups: one group consists of A. hatchetti, A. triangularis, and 3 Korean isolates (YM-2, -3, -4), and the other group consists of A. culbertsoni, A. polyphaga, HOV, and YM-5.
Topics: Acanthamoeba; Animals; Korea; Polymerase Chain Reaction; Random Amplified Polymorphic DNA Technique
PubMed: 8591012
DOI: 10.3347/kjp.1995.33.4.341 -
Microbial Ecology May 1994Adaptation of amoebae to four cooling tower Biocides, which included a thiocarbamate compound, tributyltin neodecanoate mixed with quaternary ammonium compounds...
Adaptation of amoebae to four cooling tower Biocides, which included a thiocarbamate compound, tributyltin neodecanoate mixed with quaternary ammonium compounds (TBT/QAC), another QAC alone, and an isothiazolin derivative, was studied. Previously we found that amoebae isolated from waters of cooling towers were more resistant to cooling tower biocides than amoebae from other habitats. Acanthamoeba hatchetti and Cochliopodium bilimbosum, obtained from American Type Culture Collection and used in the previous studies, were tested to determine whether they could adapt to cooling tower Biocides. A. hatchetti was preexposed to subinhibitory concentrations of the four Biocides for 72h, after which they were tested for their resistance to the same and other biocides. C. bilimbosum was exposed to only two biocides, as exposure to the other two was lethal after 72 h. Preexposure to the subinhibitory concentrations of the Biocides increased the resistance of the amoebae, as indicated by a significant increase in the minimum inhibitory concentration (up to 30-fold). In addition, cross-resistance was also observed, i.e., exposure to one biocide caused resistance to other biocides. These results show that amoebae can adapt to biocides in a short time. The phenomenon of cross-resistance indicates that regularly alternating biocides, as is done to control microbial growth in cooling towers, may not be effective in keeping amoeba populations in check. On the contrary, exposure to one biocide may boost the amoebae's resistance to a second biocide before the second biocide is used in the cooling tower. Since amoebae may harbor Legionella, or alone cause human diseases, these results may be important in designing effective strategies for controlling pathogens in cooling towers.
PubMed: 24190342
DOI: 10.1007/BF00182412 -
Applied and Environmental Microbiology Oct 1993Two species of amoebae were isolated from the cooling tower of an air-conditioning system and examined for effects of exposure to four cooling tower biocides, a...
Two species of amoebae were isolated from the cooling tower of an air-conditioning system and examined for effects of exposure to four cooling tower biocides, a thiocarbamate compound, tributyltin neodecanoate mixed with quaternary ammonium compounds, another quaternary ammonium compound alone, and an isothiazolin derivative. The amoebae isolated were Acanthamoeba hatchetti and a Cochliopodium species. Two other amoeba cultures, an A. hatchetti culture and Cochliopodium bilimbosum, were obtained from the American Type Culture Collection (ATCC) and were also tested. The cooling tower isolates were more resistant to most of the biocides than the ATCC isolates were. The isothiazolin derivative was the least inhibitory to all four amoeba isolates, and tributyltin neodecanoate mixed with quaternary ammonium compounds was the most inhibitory to three of the four isolates. After exposure to lower concentrations of the biocides, including for one strain the manufacturer's recommended concentration of one biocide, the cooling tower amoeba populations increased significantly compared with unexposed controls, whereas the ATCC isolates were not stimulated at any of the concentrations tested. In some cases, concentrations which stimulated cooling tower amoebae inhibited the growth of the ATCC isolates. These results suggest that cooling tower amoebae may adapt to biocides, underscoring the need to use freshly isolated cooling tower organisms rather than organisms from culture collections for testing the efficacy of such biocides. The stimulatory effect of biocides on amoeba populations is an alarming observation, since these organisms may be reservoirs for legionellae. Biocides used to control microbial growth may actually enhance populations of host organisms for pathogenic bacteria.
Topics: Acanthamoeba; Air Conditioning; Amoeba; Animals; Dimethyldithiocarbamate; Disinfectants; Legionella; Quaternary Ammonium Compounds; Thiazoles; Trialkyltin Compounds; Water; Water Microbiology
PubMed: 8250551
DOI: 10.1128/aem.59.10.3245-3249.1993 -
Journal of Ophthalmic Nursing &... 19931. Acanthamoeba keratitis is an uncommon but increasingly prevalent infection with the potential to cause severe ocular damage. Acanthamoeba is a nonflagellated...
1. Acanthamoeba keratitis is an uncommon but increasingly prevalent infection with the potential to cause severe ocular damage. Acanthamoeba is a nonflagellated free-living amoeba that is ubiquitous in the environment. The most common type is A castellani, but A polyphagia, A rhysodes, A culbertson, and A hatchetti have been isolated from infected eyes. 2. Clinical features include foreign body sensation, blurred vision, tearing, and photophobia. There is minimal pain in the early stages of infection, but severe pain is a manifestation of the advanced stages of the disease. 3. The incidence of Acanthamoeba keratitis appears to be decreasing because of increased awareness, but education emphasizing proper lens sterilization is essential and should be carried out when contact lenses are first dispensed.
Topics: Acanthamoeba Keratitis; Humans; Primary Prevention; Prognosis
PubMed: 8158668
DOI: No ID Found