Keratitis by Acanthamoeba triangularis: report of cases and characterization of isolates

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: keratopathogenicity of isolates from domestic tap water in Korea

In a previous study, we reported on the contamination rate of free living amoeba, including Acanthamoeba, isolated from contact lens storage cases (CLSC) and domestic tap water in Korea. In an effort to evaluate the potential kerato-pathogenicity of 5 isolates from CLSC and 17 isolates from domestic tap water, we have conducted an investigation into the morphological features, mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) phenotypes, 18S rDNA sequences, and drug sensitivities of these isolates, and have compared the results with those of 20 amoebic keratitis (AK) isolates from Korea, as well as 14 reference strains. Cysts from 22 isolates obtained from CLSC and domestic tap water showed typical characteristics of morphological group 2. A total of three and five mtDNA RFLP patterns generated by EcoRI were found in 5 of the isolates from CLSC and 17 of the isolates from domestic tap water, respectively. The mtDNA RFLP patterns of four of the five isolates from the CLSC were found to be identical to those of the isolates from domestic tap water of students who had contaminated CLSC. The majority had mtDNA RFLP patterns identical to those of AK isolates in Korea. The results of 18S rDNA sequencing analysis were also shown to coincide with the results of mtDNA RFLP analysis. KA/WP12 was determined to be profoundly sensitive to chlorhexidine (MCC; 6.25microg/ml), and KAWP2 was the most sensitive strain to polyhexamethylene biguanide (PHMB) (MCC; 4.69microg/ml). Some difference in the cytopathic effects of isolates against human corneal epithelial cells was observed according to their mtDNA genotypes. In conclusion, domestic tap water may constitute a source of Acanthamoeba contamination of CLSC, and most isolates from CLSC and domestic tap water appear to be potentially keratopathogenic.     

Evaluation of taxonomic validity of four species of Acanthamoeba: A. divionensis, A. paradivionensis, A. mauritaniensis, and A. rhysodes, inferred from molecular analyses

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.     

A riboprinting scheme for identification of unknown Acanthamoeba isolates at species level

We describe a riboprinting scheme for identification of unknown Acanthamoeba isolates at the species level. It involved the use of 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 RFCP of 17 strains in group II by Dde I, Taq I and Hae III were classified into: (1) four taxa that were identifiable at the species level, (2) a subgroup of 4 taxa and a pair of 2 taxa that were identical with each other, and (3) a species complex of 7 taxa assigned to A. castellanii complex that were closely related. These results were consistent with those obtained by 18s rDNA sequence analysis. This approach provides an alternative to the rDNA sequencing for rapid identification of a new clinical isolate or a large number of environmental isolates of Acanthamoeba.     

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

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.     

Comparison of specific activity and cytopathic effects of purified 33 kDa serine proteinase from Acanthamoeba strains with different degree of virulence

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.     

Pathogenic free-living amoebae in Korea

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.     

Molecular characterization of bacterial endosymbionts of Acanthamoeba isolates from infected corneas of Korean patients

The endosymbionts of 4 strains of Acanthamoeba (KA/E9, KA/E21, KA/E22, and KA/E23) isolated from the infected corneas of Korean patients were characterized via orcein stain, transmission electron microscopic examination, and 16S rDNA sequence analysis. Double membrane-bound, rod-shaped endosymbionts were distributed randomly throughout both the trophozoites and cysts of each of Acanthamoeba isolates. The endosymbionts of KA/E9, KA/E22, and KA/E23 were surrounded by electron-translucent areas. No lacunae-like structures were observed in the endosymbionts of KA/E21, the bacterial cell walls of which were studded with host ribosomes. Comparative analyses of the 16S rDNA sequences showed that the endosymbionts of KA/E9, KA/E22 and KA/E23 were closely related to Caedibacter caryophilus, whereas the KA/E21 endosymbiont was assigned to the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum. In the 4 strains of Acanthamoeba, the hosts of the endosymbionts were identified as belonging to the Acanthamoeba castellanii complex, which corresponds to the T4 genotype. Acanthamoeba KA/E21 evidenced characteristics almost identical to those of KA/E6, with the exception of the existence of endosymbionts. The discovery of these endosymbionts from Acanthamoeba may prove essential to future studies focusing on interactions between the endosymbionts and the amoebic hosts.     

Phylogenetic relationships among Acanthamoeba spp. based on PCR-RFLP analyses of mitochondrial small subunit rRNA gene.

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.     

Novel bacterial endosymbionts of Acanthamoeba spp. related to the Paramecium caudatum symbiont Caedibacter caryophilus

Acanthamoebae are increasingly being recognized as hosts for obligate bacterial endosymbionts, most of which are presently uncharacterized. In this study, the phylogeny of three Gram-negative, rod-shaped endosymbionts and their Acanthamoeba host cells was analysed by the rRNA approach. Comparative analyses of 16S rDNA sequences retrieved from amoebic cell lysates revealed that the endosymbionts of Acanthamoeba polyphaga HN-3, Acanthamoeba sp. UWC9 and Acanthamoeba sp. UWE39 are related to the Paramecium caudatum endosymbionts Caedibacter caryophilus, Holospora elegans a n d Holospora obtusa . With overall 16S rRNA sequence similarities to their closest relative, C. caryophilus , of between 87% and 93%, these endosymbionts represent three distinct new species. In situ hybridization with fluorescently labelled endosymbiont-specific 16S rRNA-targeted probes demonstrated that the retrieved 16S rDNA sequences originated from the endosymbionts and confirmed their intracellular localization. We propose to classify provisionally the endosymbiont of Acanthamoeba polyphaga HN-3 as ' Candidatus Caedibacter acanthamoebae', the endosymbiont of Acanthamoeba sp. strain UWC9 as ' Candidatus Paracaedibacter acanthamoebae' and the endosymbiont of Acanthamoeba sp. strain UWE39 as ' Candidatus Paracaedibacter symbiosus'. The phylogeny of the Acanthamoeba host cells was analysed by comparative sequence analyses of their 18S rRNA. Although Acanthamoeba polyphaga HN-3 clearly groups together with most of the known Acanthamoeba isolates (18S rRNA sequence type 4), Acanthamoeba sp. UWC9 and UWE39 exhibit < 92% 18S rRNA sequence similarity to each other and to other Acanthamoeba isolates. Therefore, we propose two new sequence types (T13 and T14) within the genus Acanthamoeba containing, respectively, Acanthamoeba sp. UWC9 and Acanthamoeba sp. UWE39.     

Phospholipase activities in clinical and environmental isolates of Acanthamoeba

The pathogenesis and pathophysiology of Acanthamoeba infections remain incompletely understood. Phospholipases are known to cleave phospholipids, suggesting their possible involvement in the host cell plasma membrane disruption leading to host cell penetration and lysis. The aims of the present study were to determine phospholipase activities in Acanthamoeba and to determine their roles in the pathogenesis of Acanthamoeba. Using an encephalitis isolate (T1 genotype), a keratitis isolate (T4 genotype), and an environmental isolate (T7 genotype), we demonstrated that Acanthamoeba exhibited phospholipase A(2) (PLA(2)) and phospholipase D (PLD) activities in a spectrophotometry-based assay. Interestingly, the encephalitis isolates of Acanthamoeba exhibited higher phospholipase activities as compared with the keratitis isolates, but the environmental isolates exhibited the highest phospholipase activities. Moreover, Acanthamoeba isolates exhibited higher PLD activities compared with the PLA(2). Acanthamoeba exhibited optimal phospholipase activities at 37℃ and at neutral pH indicating their physiological relevance. The functional role of phospholipases was determined by in vitro assays using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. We observed that a PLD-specific inhibitor, i.e., compound 48/80, partially inhibited Acanthamoeba encephalitis isolate cytotoxicity of the host cells, while PLA(2)-specific inhibitor, i.e., cytidine 5'-diphosphocholine, had no effect on parasite-mediated HBMEC cytotoxicity. Overall, the T7 exhibited higher phospholipase activities as compared to the T4. In contract, the T7 exhibited minimal binding to, or cytotoxicity of, HBMEC.     

Immunological inter-strain crossreactivity correlated to 18S rDNA sequence types in Acanthamoeba spp

Various species of the genus Acanthamoeba have been described as potential pathogens; however, differentiation of acanthamoebae remains problematic. The genus has been divided into 12 18S rDNA sequence types, most keratitis causing strains exhibiting sequence type T4. We recently isolated a keratitis causing Acanthamoeba strain showing sequence type T6, but being morphologically identical to a T4 strain. The aim of our study was to find out, whether the 18S rDNA sequence based identification correlates to immunological differentiation. The protein and antigen profiles of the T6 isolate and three reference Acanthamoeba strains were investigated using two sera from Acanthamoeba keratitis patients and one serum from an asymptomatic individual. It was shown, that the T6 strain produces a distinctly different immunological pattern, while patterns within T4 were identical. Affinity purified antibodies were used to further explore immunological cross-reactivity between sequence types. Altogether, the results of our study support the Acanthamoeba 18S rDNA sequence type classification in the investigated strains.     

Diversity of halophilic archaea from six hypersaline environments in Turkey

The diversity of archaeal strains from six hypersaline environments in Turkey was analyzed by comparing their phenotypic characteristics and 16S rDNA sequences. Thirty-three isolates were characterized in terms of their phenotypic properties including morphological and biochemical characteristics, susceptibility to different antibiotics, and total lipid and plasmid contents, and finally compared by 16S rDNA gene sequences. The results showed that all isolates belong to the family Halobacteriaceae. Phylogenetic analyses using approximately 1,388 bp comparisions of 16S rDNA sequences demonstrated that all isolates clustered closely to species belonging to 9 genera, namely Halorubrum (8 isolates), Natrinema (5 isolates), Haloarcula (4 isolates), Natronococcus (4 isolates), Natrialba (4 isolates), Haloferax (3 isolates), Haloterrigena (3 isolates), Halalkalicoccus (1 isolate), and Halomicrobium (1 isolate). The results revealed a high diversity among the isolated halophilic strains and indicated that some of these strains constitute new taxa of extremely halophilic archaea.     

Pathogenicity of Korean isolates of Acanthamoeba by observing the experimental infection and zymodemes of five isoenzymes.

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.     

大连海域沉积物中可培养海洋链霉菌活性及其多样性

以大肠杆菌、金黄色葡萄球菌和尖孢镰刀枯萎病菌作为测试靶目标,采用9种分离培养基从大连海域13个不同采样点的海洋沉积物样品中分离到165株海洋链霉菌。从165株海洋放线菌中筛选到对金黄色葡萄球菌具有抑制活性的菌株85株,占总菌株数的51.5%;对大肠杆菌具有抑制活性的菌株27株,占总菌株数的16.4%;对尖孢镰刀枯萎病菌具有抑制活性的菌株仅有6株,占总菌株数的3.6%。因此,海洋链霉菌的活性更多地表现为对细菌的抗性,尤其对革兰氏阳性细菌具有更高的抑制活性。对其中具有抑制活性或形态独特的菌株进行了16S r DNA序列分析,并构建系统发育树,显示活性海洋链霉菌具有丰富的种类多样性和广谱抗菌活性。同种海洋链霉菌与土壤链霉菌活性比较结果也表明,海洋链霉菌多表现抗革兰氏阳性细菌活性。     

Correlations between morphological, molecular biological, and physiological characteristics in clinical and nonclinical isolates of Acanthamoeba spp

Eleven Acanthamoeba isolates, obtained from Acanthamoeba keratitis patients, from contact lens cases of non-Acanthamoeba keratitis patients, from asymptomatic individuals, from necrotic tissue, and from tap water and two reference strains were investigated by morphological, molecular biological, and physiological means in order to discriminate clinically relevant and nonrelevant isolates. All clinically relevant isolates showed Acanthamoeba sp. group II morphology. 18S ribosomal DNA sequencing revealed sequence type T4 to be the most prevalent group among the isolates and also the group recruiting most of the pathogenic strains. Interestingly, within T4 the strains of no clinical relevance clustered together. Moreover, physiological properties appeared to be highly consistent with initial pathogenicity and with sequence clustering. Altogether, the results of our study indicate a correlation between the phylogenetic relationship and pathogenicity.     

Subgenus Systematics of Acanthamoeba: Four Nuclear 18S rDNA Sequence Types

ABSTRACT Classification of Acanthamoeba at the subgenus level has been problematic, but increasing reports of Acanthamoeba as an opportunistic human pathogen have generated an interest in finding a more consistent basis for classification. Thus, we are developing a classification scheme based on RNA gene sequences. This first report is based on analysis of complete sequences of nuclear small ribosomal subunit RNA genes ( Rns ) from 18 strains. Sequence variation was localized in 12 highly variable regions. Four distinct sequence types were identified based on parsimony and distance analyses. Three were obtained from single strains: Type T1 from Acanthamoeba castellanii V006, T2 from Acanthamoeba palestinensis Reich, and T3 from Acanthamoeba griffini S-7. T4, the fourth sequence type, included 15 isolates classified as A. castellanii, Acanthamoeba polyphaga, Acanthamoeba rhysodes , or Acanthamoeba sp., and included all 10 Acanthamoeba keratitis isolates. Interstrain sequence differences within T4 were 0%–4.3%, whereas differences among sequence types were 6%–12%. Branching orders obtained by parsimony and distance analyses were inconsistent with the current classification of T4 strains and provided further evidence of a need to reevaluate criteria for classification in this genus. Based on this report and others in preparation, we propose that Rns sequence types provide the consistent quantititive basis for classification that is needed.     

Diversity of Streptomyces spp. in Eastern Himalayan region - computational RNomics approach to phylogeny

Isolation and characterization of actinomycetes from soil samples from altitudinal gradient of North-East India were investigated for computational RNomics based phylogeny. A total of 52 diverse isolates of Streptomyces from the soil samples were isolated on four different media and from these 6 isolates were selected on the basis of cultural characteristics, microscopic and biochemical studies. Sequencing of 16S rDNA of the selected isolates identified them to belong to six different species of Streptomyces. The molecular morphometric and physico-kinetic analysis of 16S rRNA sequences were performed to predict the diversity of the genus. The computational RNomics study revealed the significance of the structural RNA based phylogenetic analysis in a relatively diverse group of Streptomyces.