Parasitologia Hungarica 28. (Budapest, 1995)

Parasitologia Hungarica 28. (Budapest, 1995)

© Hungarian Natural History Museum

Hungarian Society of Parasitologists

Parasit, hung., 28: 5-12, 1995

Isolation and restriction enzyme analysis of

mitochondrial DNA (mtDNA) of Hungarian

Acanthamoeba isolates from human eye

infection and from the environment

Zsuzsanna SZÉNÁSI 1 , Takuro ENDO 2 , Kenji VAGUA 2 ,

Edit URBAN 1 , Mihály VÉGH 3 and Erzsébet NAGY 1


Department of Clinical Microbiology, Albert Szent-Györgyi Medical

University, H-670J Szeged, P.O. Box 482, Hungary


Department of Parasitology, National Institute of Health,

Shinagawa-Ku, Tokyo 141, Japan


^Department of Ophthalmology, Albert Szent- Györgyi Medical

University, H-6720 Szeged, Korányi fasor 7-11, Hungary

(Received 18 June, 1995)

Abstract: Restriction fragment length polymorphism (RFLP) analysis was used

for the typing of Acanthamoeba isolates from the contact lens container and corneal

scrapings of a patient with keratitis (CI) and for typing isolates from the

environment (Dun, Mos). The Acanthamoebae were isolated on agar plates covered

with a suspension of Enterobacter aerogenes. The isolates were cloned by

micromanipulation, then cultured and maintained on agar plates spread with a

layer of heat-treated Escherichia coli DH1. The cloned isolates were adapted

and cultured axenically. The mitochondrial DNA (mtDNA) was prepared by alkaline

lysis and digested by Bg\ II and £coR I endonucleases. The three isolates

have distinct RFLP phenotypes. On the basis of the fragment sizes obtained with

two restriction enzymes, the mtDNA genotypes of the three Hungarian isolates

are comparable to those of human isolates from different countries. As all strains

of Acanthamoeba may have pathogenic potential, the mtDNA RFLP phenotype

may be a useful aid in the definition of their pathogenic potential.

Key words: Acanthamoeba, eye infection, RFLP analysis, mtDNA


Infections caused by small free-living amoebae are generally not very well known or

recognized clinically. Most laboratories are unfamiliar with the diagnostic methods of such

infections and, therefore, do not use them on a routine basis. Free-living Acanthamoeba

spp are common predators of bacteria. They are ubiquitous in nature. Dry cysts can survive

for several years and are regularly isolated from natural waters, inadequately treated

swimming pools, soil, freshwater fishes, brackish water, ocean sediments, dust, air,

and even from bottled mineral water (Sawyer et al. 1977, Brown et al. 1982, Franke

and Mackievicz 1982,Kyle and Noblet 1986,). This normally free-living organism

occasionally appears as an opportunist parasite of man (Cerva 1981). Acanthamoeba

may sometimes occur as a commensalist in the nasopharynx of apparently healthy

normal humans (Rivera et al. 1984) and of patients with possible virus infection, and

it is not infrequently found in tissue cultures used for virus isolation from nasal swabs

(Griffin 1978). Acanthamoeba spp have been reported from the central nervous system

(CNS), corneal ulcers, the genitourinary tract and many other sites of the human body

(Wang and Feldman 1967, Carter 1972, Ashton and Stamm 1975, Lund 1978, Key et

al. 1980, Cerva 1981, Samples et al. 1984, Cohen et al. 1985, Moore et al. 1986,

Epstein et al. 1986, Ormerod and Smith 1986, Cohen et al. 1987, Mathers et al. 1987,

Moore et al. 1987, Koenig et al. 1987, Silvany et al. 1987, Stehr-Green et al. 1987,

Lindquist 1988, Brandt 1989, Stehr-Green 1989). There are a number of published

cases that emphasize the need for clinicians to consider acanthamoebic infection in the

differential diagnosis of eye infections that fail to respond to antibacterial, antifungal

or antiviral therapy (Ma et al. 1981, Hirst et al. 1984, Cohen et al. 1985, Moore et

al. 1985, Mannis et al. 1986). These infections are often due to direct eye exposure to

contaminated materials or solutions.

We have isolated Acanthamoeba spp from a commercial contact lens cleaning-disinfecting

solution, from the corneal scrapings of a patient with severe eye soreness, and from

the environment.

The taxonomy of these small amoeba species is not well established. Consequently,

little is known about the relationship between pathogenic and nonpathogenic strains. Several

studies report on the analysis of mtDNA variation in members of the genus Acanthamoeba

as an aid in taxonomy (Bogler et al. 1983, Byers et al. 1983, Costas et al. 1983, Yagita

and Endo 1990). This method has proved to be a useful approach to studying the

evolutionary relationship among closely related organisms (Brown and Simpson 1981,

Yonekawa et al. 1981). We used the RFLP analysis (Yagita and Endo 1990) for the typing

of Acanthamoeba isolates from man and from the environment (CI, Dun, Mos) and for

comparing them with human isolates from different countries other than Hungary.


Isolation and culture of Acanthamoeba

Preparation of agar plates: Non-nutrient agar plates are prepared by pouring 12-15

ml of sterile 1.5% agar in water, into 90 mm diameter Petri dishes. When the agar has

hardened, the plates are stored at 4 0

C. The agar plates can be safely stored for 2-3 weeks.

Prior to use, the plates are placed in a 37 °C incubator for 30 min. A thin suspension of

heat-treated (60 °C for 60 min) Enterobacter aerogenes or Escherichia coli DH1 that does

not contain bacterial plasmid is spread onto the surface of the agar. The agar plates coated

with bacteria are dried in air under axenic conditions.

PYG medium (pH 7) for axenic culture: Proteose-peptone (Difco) 10 g, Yeast extract

(Difco) 10 g, NaCl 5 g, 0.5 M Na2HP04 10 ml, 0.5 M KH2PO4 10 ml, distilled water

970 ml, autoclaved at 121 °C for 15 min. The following reagents are added aseptically:

50% glucose in water 10 ml (Millipore filtered), Penicillin K. 200,000 U/l, Streptomycin

Sulfate 200,000 ug/1.

Inoculation of plates and observation: A small volume of water (preferably the

sediment of the centrifuged water) or medical samples are placed on the center of two agar

plates coated with bacteria and incubated parallel at 25-30 °C and 42-45 °C, respectively.

Observations are made daily with the unaided eye and under a microscope (preferably an

inverted microscope) for up to 7 days. If amoebae are present, they will feed on bacteria

and multiply, then move away centripetally from the area of inoculation. After several days

of incubation, the amoebae will begin to encyst. A small piece of agar is excised with a

sterile lancet from an area positive for amoebae, and transferred upside down onto a fresh

agar plate precoated with bacteria.

Table 1

Sources of the isolated strains






Contact lens container and corneal scrapings of a patient with severe eye soreness.

Her symptoms appeared when using daily-wear soft contact lenses during her

vacation at Lake Balaton

The root of a moss

The River Danube

Cloning and axenization - micromanipulation method: A single cell (preferably cyst)

is collected using a sterile microcapillary tube and transferred to a fresh agar plate precoated

with bacteria. The dish is then placed into an incubator.

For axenization, the cysts from a clonal culture are kept in 0.1 N HCl at room

temperature overnight. They are washed several times with an excess amount of sterile

water to neutralize the pH of the suspension, and are inoculated into an axenic culture

medium (PYG medium) containing antibiotics. The cloned isolates were adapted and

cultured axenically at 26 °C in PYG medium for large-scale sampling of the organisms.

All the procedures should be carried out in a laminar flow cabinet or similar clean

environment to eliminate all risk of airborne contaminants.

Restriction fragment length polymorphism (RFLP) analysis

Mitochondrial DNA from the isolates of Acanthamoeba was obtained by the method

of alkaline lysis. Aliquots of midlog phase amoebae (5x10 cells) grown in PYG medium

were collected and washed three times in phosphate buffered saline (pH 7.2) by centrifuging

at 300 g for 5 min. The pelleted amoebae were washed again three times in a 1.5 ml

Eppendorf centrifuge tube in TES buffer (50 mM Tris HCl, 10 mM ethylenediamine

tetraacetic acid (EDTA), 50 mM sucrose, pH 8.0).

The pellet was resuspended in 100 pi of chilled TES and added to 200 pi of freshly

prepared 1% sodium dodecyl sulfate solution in 0.2 N NaOH. The suspension was gently

mixed by inverting the tubes and incubated at 0 °C for 5 min. Then, 150 pi of chilled 3

M potassium acetate buffer was added to the suspension. The suspension was mixed and

incubated for 30 min at 0°C. After centrifugádon (12,000 g for 10 min at 4 °C), the

supernatant fluid was transferred to a fresh Eppendorf tube and an equal volume of

phenol/chloroform (1:1) solution was added. The supernatant fluid was collected following

centrifugádon at 12,000 g for 10 min at 4 °C. The DNA fraction was sedimented by adding

1.0 ml of cold absolute ethanol and 35 pi of 3 M sodium acetate solution at -70 °C. The

supernatant fluid was removed following centrifugádon at 18,000 g for 15 min and the

sample was dried with a centrifugal evaporator (Tomy, Tokyo) at 230 g for 2-3 min at 42 °C.

The sediment was dissolved in 30-50 pi of TE buffer (5 mM Tris HCl pH 8.0, 1 mM

EDTA) and stored at -20 °C until used.

Approximately, 0.15 pg of the mtDNA sample was incubated overnight with 5-10

units of restriction enzymes Bgl II and EcoK I at 37 °C in a total volume of 50 pi using

buffers specified by the enzyme supplier. The enzymes were purchased from Takara Shuzo

(Kyoto, Japan).

The digests were electrophoresed in 0.7% agarose gel at a constant voltage of 3 V/cm

for 6 h in TBE buffer (89 mM Tris-borate, 89 mM boric acid, 2 mM EDTA).

The gel was stained with ethidium bromide and observed with a short wave ultraviolet

transilluminator. The Hae III digests of O XI74 phage and the Sal I and Hind III digests

of Lambda phage were included in each gel run as size standards.

Table 2

Estimates of mtDNA restriction fragment sizes for 2 endonucleases

Endonuclease Fragments CI Dun Mos

Bgl II 1 20,100 9,200 11,300

2 6,300 6,300 10,200

3 6,000 5,700 6,600

4 5,400 5,500 4,500

5 2,000 4,500 3,600

6 800 3,100 2,300

7 2,500 1,400

EcoK I 1 10,500 13,200 11,700

2 8,500 12,700 10,700

3 7,500 8,600 7,600

4 6,600 5,000 6,900

5 4,300 1,800 3,200

6 3,300 1,400 2,500


Morphologically, all the three isolated strains were identified as Acanthamoeba spp

mainly on the basis of cyst structure (Fig. 1. A, B, C). The endocysts are usually stellated

and the ectocysts (the outer wall) are wrinkled. The vegetative form is larger, nuclei and

granular cytoplasm can be seen. Comparing A, B and C, no morphologic distinction can

be made. For the RFLP analysis, the amoebae were cultured on agar plates covered with

plasmid free E. coli DH1 to prevent contamination of the DNA samples with bacterial

plasmids. Mitochondrial DNA from the isolates of Acanthamoeba was obtained by alkaline

lysis. Using this method, mtDNA could be isolated from whole amoebae of monoxenic

cultures without prior purification of the mitochondria. Mitochondrial DNA from the

isolates was digested with two six base recognition restriction enzymes: Bgl II and EcoK I

which showed the highest enzymatic activity at 37 °C (pH 7.5) and produced 6 7 fragments

(Fig. 1). Fragment sizes obtained with two restriction enzymes are given in Table 2. In a

comparison of our results with those reported by Yagita and Endo (1990), the RFLP

phenotype of CI is comparable to JAC/E4 from 6 countries excluding Hungary, Dun is

comparable to Ma from 9 countries other than Hungary, and Mos is comparable to NZAU

from New Zealand (unpublished data).

0>X174 Lambda CI MOS Dun CI MOS Dun lambda

Hae\\\Sal\ Bgl II EcoRl HindlU

Fig. 1. Morphologic features of the cysts and the vegetative forms of the isolates on agar

plates. A clinical isolate (Cl); B — isolate from the moss (Mos); C — isolate from the river

Danube; D - Comparison of agarose gel electrophoretic patterns of mtDNA for Bgl II

(lane 3: CI, lane 4: Mos, lane 5: Dun) and for EcoK 1 (lane 6: CI, lane

7: Mos, lane 8: Dun) digests.

Note the usually stellated endocyst and wrinkled outer wall of cysts. The vegetative forms are larger, nuclei and

granular cytoplasm can be seen. Comparing A, B and C, no morphologic distinction can be made.


As Acanthamoeba keratitis is not a notifiable disease, its incidence is unknown.

Although the disease is relatively rare, its incidence may be higher than expected from the

reports published.

We have isolated an Acanthamoeba strain (CI) from the contact lens container and corneal

scrapings of a patient with eye soreness. Morphologically, the isolate was undistinguishable

from the Acanthamoeba strains isolated from a moss (Mos) or from the river Danube (Dun).

Using RFLP analysis, our pathogenic strain (CI) and the strains isolated from the environment

were found to be similar to the acanthamoebae isolated from human patients in different

countries. The JAC\E4 and Ma strains reported by Yagita and Endo (1990) were isolated from

human eye infection. Thus, RFLP phenotype analysis of the mtDNA may be a useful tool for

the taxonomic classification of free-living amoeba species and perhaps also for the determination

of their pathogenic potential.

The growing use of contact lenses increses the number of persons at risk of

Acanthamoeba keratitis. Therefore, it is essential that ophthalmologists carefully explain

the procedures of proper lens cleaning and care to their patients.

Szénási Zs., Endo T., Yagita K., Urban E., Végh M. és Nagy E.: Szemészeti

anyagból és a környezetből Magyarországon izolált acanthamoebák jellemzése a

mitokondriális DNS (mt DNS) restrikciós enzimes emésztésével

A szerzők keratitises beteg kontaktlencse tárolójából és szaruhártya törmelékéből

kitenyésztett Acanthamoeba-X.(Cl)ésakörnyezetből származó Acanthamoeba izolátumokat

(Dun, Mos) a mtDNS restrikciós enzimes emésztésével tipizálták. Az acanthamoebák

tenyésztésére Enterobacter aerogenes szuszpenzióval fedett agar lemezeket használtak.

Az izolátumokat mikromanipulációs módszerrel kiónozták, majd hőkezelt Escherichia

coli DH1 szuszpenzióval fedett agar lemezeken szaporították, ezt követően pedig

axenikus tenyésztéshez szoktatták. A mtDNS-t alkalikus lízissel nyerték ki és a Bgl II

és az EcoRI endonukleázzal emésztették. A restrikciós emésztéssel keletkezett

fragmentek (RF) méreteit (RFL) összehasonlították más országokból származó

Acanthamoeba izolátumok fragment méreteivel. Eredményeik szerint a fragmentek

méretei alapján nemcsak csoportosíthatók a földrajzilag eltérő származású izolátumok,

hanem lehetséges patogenitásukra is következtetni lehet.


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