Transmission electron microscopy of an Acanthamoeba isolate (KA/L5) from a contact lens case revealed bacterial endosymbionts within cytoplasm of the amoebae. The Acanthamoeba isolate belonged to the morphological group II. Based on the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) of 18S ribosomal RNA coding DNA (rDNA), the isolate was identified as A. lugdunensis. Strain typing by isoenzyme analysis using isoelectric focusing (IEF) and mitochondrial (Mt) DNA RFLP revealed that the isolate was closely related with KA/L1, the most predominant type of isolates from contact lens storage cases, KA/E2, a clinical isolate, KA/W4, previously reported to host endosymbionts, and L3a strains of A. lugdunensis. The endosymbionts were similar to those of KA/W4 in aspects that they were randomly distributed in both trophozoites and cysts, and were rod-shaped bacteria measuring approximately 1.38 x 0.50 microns. But the number of endosymbionts per amoeba was significantly lower than that of KA/W4. They were neither limited by phagosomal membranes nor included in lacunaelike structure.
Acanthamoeba/microbiology* ; Acanthamoeba/cytology ; Animal ; Bacteria/isolation & purification* ; Colony Count, Microbial ; Contact Lenses* ; Cytoplasm/microbiology ; Symbiosis*

OBJECTIVETo determine whether Acanthamoeba polyphaga could affect the survival and growth of Vibrio cholerae O139 in low temperature.

METHODSV. cholerae O139 was co-cultured with the Acanthamoeba polyphaga to be examined on its intracellular growth and survival rate within cysts at low temperature, using methods as Gram-staining, electron microscope and passage culture.

RESULTSV. cholerae O139 were observed to enter into the trophozoites and grow the within the vacuoles after 8 hour incubation with Acanthamoeba polyphaga. The germs survived in the vacuole and/or endo-layer of wall and could be re-isolated from the excystment of Acanthamoeba polyphaga. At 30 degrees C, V. cholerae O139 could survive for 120 days with the amoeba while less than 45 days in PAS. At 4 degrees C, the number of viable bacteria decreased and reached undetectable levels for both study and control groups after a 30-day incubation. V. cholerae O139 could be re-isolated from the 30-, 45-, 60- and 75-day's infected cysts after excystment. However the ability of excystment for 90-day's infected cysts decreased and V. cholerae O139 within the cyst could not be isolated again because the amoebae had lysed.

CONCLUSIONThese findings indicated that V. cholerae O139 could grow within Acanthamoeba polyphaga and the survival time could be increased in the cysts at low temperature. It seemed that Acanthamoeba can provide an environmental reservoir for V. cholerae O139.

Acanthamoeba ; microbiology ; Bacterial Capsules ; Colony Count, Microbial ; Temperature ; Vibrio cholerae ; growth & development

The existence of symbiotic relationships between Acanthamoeba and a variety of bacteria is well-documented. However, the ability of Acanthamoeba interacting with host bacterial pathogens has gained particular attention. Here, to understand the interactions of Escherichia coli K1 and E. coli K5 strains with Acanthamoeba castellanii trophozoites and cysts, association assay, invasion assay, survival assay, and the measurement of bacterial numbers from cysts were performed, and nonpathogenic E. coli K12 was also applied. The association ratio of E. coli K1 with A. castellanii was 4.3 cfu per amoeba for 1 hr but E. coli K5 with A. castellanii was 1 cfu per amoeba for 1 hr. By invasion and survival assays, E. coli K5 was recovered less than E. coli K1 but still alive inside A. castellanii. E. coli K1 and K5 survived and multiplied intracellularly in A. castellanii. The survival assay was performed under a favourable condition for 22 hr and 43 hr with the encystment of A. castellanii. Under the favourable condition for the transformation of trophozoites into cysts, E. coli K5 multiplied significantly. Moreover, the pathogenic potential of E. coli K1 from A. castellanii cysts exhibited no changes as compared with E. coli K1 from A. castellanii trophozoites. E. coli K5 was multiplied in A. castellanii trophozoites and survived in A. castellanii cysts. Therefore, this study suggests that E. coli K5 can use A. castellanii as a reservoir host or a vector for the bacterial transmission.
Acanthamoeba castellanii/*microbiology ; Animals ; Disease Reservoirs/*microbiology ; Disease Vectors ; Escherichia coli/growth & development/pathogenicity/*physiology ; Oocysts/microbiology ; Symbiosis/*physiology ; Trophozoites/microbiology

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.
Acanthamoeba/genetics/isolation & purification/*microbiology ; Acanthamoeba Keratitis/*microbiology/*parasitology ; Animals ; Bacteria/*genetics/isolation & purification ; Base Sequence ; Cornea/microbiology/*parasitology ; DNA, Mitochondrial/genetics ; Humans ; Korea ; Microscopy, Electron, Transmission/methods ; Oxazines/metabolism ; Phylogeny ; RNA, Ribosomal, 16S/genetics ; RNA, Ribosomal, 18S/genetics ; Symbiosis

OBJECTIVETo study the survival and developmental morphology of Parachlamydia (BN9) within Acanthamoeba.

METHODSThe morphology of BN9 within Acanthamoeba was studied by inverted phase contrast microscope, electron microscope, Gimenez and AO-staining with amoebal co-culture.

RESULTSThe endosomal maturation-blocked were formed after the egress of BN9. Two developmental stages-elementary and reticulate bodies, were both observed within the vacuoles. The reticulate bodies, multiplicated by binary fission, were located mainly within the vacuoles, while the elementary bodies can also be located in the plasma individually. The naked cluster particles were observed after the trophozoites cytolysis with Gimenez-staining. The light infectious trophozoites could encyst, and elementary bodies could survive within the mature cysts.

CONCLUSIONThe egress of BN9 could form the endosomal maturation-blocked, which was presented in two developmental stages-elementary and reticulate bodies. It exhibited the cytolysin activity that could lyse the infectious trophozoites and were expelled in the vesicles. A few light infected amoeba could encyst with survival elementary bodies in the plasma.

Acanthamoeba ; microbiology ; ultrastructure ; Animals ; Chlamydiales ; physiology ; ultrastructure ; Coculture Techniques ; Humans ; Inclusion Bodies ; ultrastructure ; Life Cycle Stages ; Microscopy, Electron

OBJECTIVETo study the survival and growth of Vibrio cholerae inside the Acanthamoeba polyphage.

METHODSSurvival and growth of Vibro cholerae O139, co-cultured with Acanthamoeba polyphaga, was observed inside the trophozoites and cysts, using Gram stain and electron microscope.

RESULTSViable O139 was observed inside the amoebal vacuoles in 24 hours. Vacuoles were filled with more bacteria along with the longer period of co-culture. The process of O139 infection with Amoebae would include uptake, formation of O139 vacuole, multiplication, trophozoites lysed and expel under electron microscopy. Some infected trophozoites could subsequently encyst and the surviving O139 could locate in the vesicles inside the cysts.

CONCLUSIONO139 might survive and multiply in the trophozoites and reside inside the cysts of Amoebae, suggesting that Acanthamoebae might serve as one of the environmental hosts of Vibro cholerae.

Acanthamoeba ; growth & development ; microbiology ; ultrastructure ; Animals ; Coculture Techniques ; Colony Count, Microbial ; Culture Media ; Vibrio cholerae O139 ; growth & development ; ultrastructure ; Water ; parasitology

Recent in vitro studies have revealed that a certain Mycobacterium can survive and multiply within freeliving amoebae. It is believed that protozoans function as host cells for the intracellular replication and evasion of Mycobacterium spp. under harmful conditions. In this study, we describe the isolation and characterization of a bacterium naturally observed within an amoeba isolate acquired from a contact lens storage case. The bacterium multiplied within Acanthamoeba, but exerted no cytopathic effects on the amoeba during a 6-year amoebic culture. Trasnmission electron microscopy showed that the bacteria were randomly distributed within the cytoplasm of trophozoites and cysts of Acanthamoeba. On the basis of the results of 18S rRNA gene analysis, the amoeba was identified as A. lugdunensis. A 16S rRNA gene analysis placed this bacterium within the genus Mycobacterium. The bacterium evidenced positive reactivity for acid-fast and fluorescent acid-fast stains. The bacterium was capable of growth on the Middlebrook 7H11-Mycobacterium-specific agar. The identification and characterization of bacterial endosymbionts of free-living protozoa bears significant implications for our understanding of the ecology and the identification of other atypical mycobacterial pathogens.
Acanthamoeba/genetics/isolation & purification/*microbiology ; Animals ; Base Sequence ; Contact Lens Solutions ; *Contact Lenses ; DNA, Mitochondrial/genetics ; Microscopy, Electron, Transmission/methods ; Mycobacterium/genetics/*isolation & purification ; Phylogeny ; Polymorphism, Restriction Fragment Length ; RNA, Ribosomal, 16S/genetics ; RNA, Ribosomal, 18S/genetics ; Symbiosis

In a previous study, we reported our discovery of Acanthamoeba contamination in domestic tap water; in that study, we determined that some Acanthamoeba strains harbor endosymbiotic bacteria, via our molecular characterization by mitochondrial DNA restriction fragment length polymorphism (Mt DNA RFLP). Five (29.4%) among 17 Acanthamoeba isolates contained endosymbionts in their cytoplasm, as demonstrated via orcein staining. In order to estimate their pathogenicity, we conducted a genetic characterization of the endosymbionts in Acanthamoeba isolated from domestic tap water via 16S rDNA sequencing. The endosymbionts of Acanthamoeba sp. KA/WP3 and KA/WP4 evidenced the highest level of similarity, at 97% of the recently published 16S rDNA sequence of the bacterium, Candidatus Amoebophilus asiaticus. The endosymbionts of Acanthamoeba sp. KA/WP8 and KA/WP12 shared a 97% sequence similarity with each other, and were also highly similar to Candidatus Odyssella thessalonicensis, a member of the alpha-proteobacteria. The endosymbiont of Acanthamoeba sp. KA/WP9 exhibits a high degree of similarity (85-95%) with genus Methylophilus, which is not yet known to harbor any endosymbionts. This is the first report, to the best of our knowledge, to show that Methylophilus spp. can live in the cytoplasm of Acanthamoeba.
Acanthamoeba/isolation & purification/*microbiology/ultrastructure ; Alphaproteobacteria/classification/genetics/*isolation & purification ; Animals ; Bacteroidetes/classification/genetics/*isolation & purification ; Cluster Analysis ; DNA, Bacterial/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Fresh Water/*parasitology ; Korea ; Methylophilus/classification/genetics/*isolation & purification ; Microscopy, Electron, Transmission ; Phylogeny ; RNA, Ribosomal, 16S/genetics ; Sequence Analysis, DNA ; *Symbiosis