Isolation and identification of Acanthamoeba strains from soil and tap water in Yanji, China
Xuan et al. Environmental Health and Preventive Medicine
Isolation and identification of Acanthamoeba strains from soil and tap water in Yanji, China
Yinghua Xuan 2
Yanqin Shen 2
Yuxi Ge 1
Gen Yan 1
Shanzi Zheng 0
0 Department of Parasitology, College of Medicine, Yanbian University , Yanji 133000, Jilin , China
1 Department of Radiology, Affiliated Hospital of Jiangnan University , No. 200 Huihe Road, Wuxi 214062, Jiangsu , China
2 Department of Basic Medicine, Medical School, Jiangnan University , 1800 Li Hu Road, Wuxi 214122, Jiangsu , China
Background: Members of the genus Acanthamoeba are widely distributed throughout the world, and some of them are considered pathogenic, as they are capable of causing corneal and central nervous system diseases. In this study, we isolated Acanthamoeba strains from soil and tap water in Yanji, China. Methods: We identified four strains of Acanthamoeba (CJY/S1, CJY/S2, CJY/S3, and CJY/W1) using mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) analysis. Nuclear 18S rDNA sequences were used for phylogenetic analysis and species identification. Results: Genotypic characterization of the isolates showed that they belonged to genotypes T4 (CJY/S1 and CJY/S2), T5 (CJY/S3), and T16 (CJY/W1). Sequence differences between CJY/S1 and Acanthamoeba castellanii Neff, CJY/S2 and Acanthamoeba KA/E7, and CJY/S3 and Acanthamoeba lenticulata 68-2 were 0.31, 0.2, and 0. 26%, respectively. 18S ribosomal deoxyribonucleic acid (rDNA) of CJY/W1 had 99% sequence identity to that of Acanthamoeba sp. U/H-C1. Strains CJY/S1 and CJY/S2, isolated from soil, had similar mtDNA RFLP patterns, whereas strain CJY/W1, isolated from tap water, displayed a different pattern. Conclusions: To the best of our knowledge, this is the first report on the identification of genotypes T4, T5, and T16 from environmental sources in Yanji, China.
Acanthamoeba; Environment; Genotype; 18S rDNA; Mitochondrial DNA RFLP
Background
Acanthamoeba species are widely distributed in the
environment: their habitats include soil, freshwater,
seawater, dust, and putrilage. Some Acanthamoeba species
can cause keratitis, granulomatous amoebic encephalitis
(GAE), pulmonary infections, cutaneous lesions,
rhinosinusitis, osteomyelitis, or diffuse inflammation [
1–3
].
Acanthamoeba keratitis can lead to scarring of the
cornea, resulting in a permanent visual impairment or
complete blindness. GAE is a central nervous system
disease that usually occurs in immunocompromised
patients, such as those with acquired immune deficiency
syndrome, systemic lupus erythematosus, and
transplanted organ, or those undergoing chemotherapy for
cancer. Nonetheless, several cases of GAE and skin
infection due to Acanthamoeba spp. have been reported in
immunocompetent individuals [
4–8
].
Eye infections have been reported to be caused by
Acanthamoeba castellanii, Acanthamoeba polyphaga,
Acanthamoeba rhysodes, Acanthamoeba culbertsoni,
Acanthamoeba lugdunensis, Acanthamoeba griffini,
Acanthamoeba hatchetti, Acanthamoeba quina,
Acanthamoeba lenticulata, and Acanthamoeba triangularis.
Central nervous system diseases have been caused by
A. castellanii, A. culbertsoni, Acanthamoeba
astronyxis, A. rhysodes, Acanthamoeba healyi, and A.
lenticulata [
6, 9–13
].
There have been few reports of Acanthamoeba spp.
isolated from environmental samples in China [
14
]. In this
study, we report the molecular biological characterization
of four strains of Acanthamoeba (CJY/S1, CJY/S2, CJY/S3,
and CJY/W1) isolated from soil and tap water in China.
We identified these species by using mitochondrial DNA
restriction fragment length polymorphism (mtDNA RFLP)
analysis and 18S rDNA sequence alignment. We found
that the three strains isolated from the soil belonged to
the morphological group II and had genotypes T4 and T5,
whereas the strain isolated from tap water belonged to the
morphological group II and had genotype T16.
Methods
Isolation and cultivation of Acanthamoeba
Samples of soil and tap water were collected in Yanji,
China. The samples were loaded onto 1.5% agar plates
covered with heat-inactivated (60 °C for 1 h) Escherichia
coli (American Type Culture Collection, ATCC 25922,
free of plasmid). The plates were incubated at 25 °C, and
growth of Acanthamoeba was observed under an
inverted microscope on a daily basis for 1 week. Each
cyst isolated with a glass capillary was inoculated on a
new agar plate and incubated for 1 week. For
axenization, a piece of agar (1 cm × 1 cm) covered with cysts
was treated with 0.1 N HCl for 24 h, washed three times
with sterile water, placed in peptone yeast glucose
medium (10 g proteose peptone, 10 g yeast extract,
10 mL of 50% glucose, 10 mL of 0.5 M Na2HPO4, and
10 mL of 0.5 M K2HPO4 in 970 mL of sterile water),
and incubated at 25 °C for 3 weeks. When most of the
amoebae reached trophozoite stage, they were harvested
and washed three times with phosphate-buffered saline.
Morphological examination (...truncated)