INTRODUCTION

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Free-living amoebae of the genus Acanthamoeba are of medical interest as the causative agents of several infections in the immunodeficient host (8) and also of a very often seriously progressing keratitis occurring predominantly in contact lens wearers. Acanthamoeba keratitis has become increasingly important within the last 10 years correlating to the growing number of contact lens wearers. Contaminated contact lens care systems usually are the first step in an Acanthamoeba keratitis pathogenesis.

Various species have been reported to be able to cause keratitis: Acanthamoeba castellanii, A. polyphaga, A. hatchetti, A. culbertsoni, A. rhysodes, A. lugdunensis, A. quina, and A. griffini (23). However, accurate species determination remains problematic. A great advantage in the differentiation of acanthamoebae was the division of the genus into three morphological groups by Pussard and Pons (22), with a majority of keratitis causing strains belonging to group II. Recently, Stothard et al. (26) identified 12 Acanthamoeba sequence types by 18S ribosomal DNA (rDNA) sequencing and, interestingly, they found evidence that most keratitis causing strains group into sequence type T4. However, a final and generally accepted system does not yet exist. Physiological characteristics such as temperature tolerance (11), growth in defined media (4), the ability to migrate in an "under-agarose" system (28), and cytopathic effects in human cell lines (3) were found to be pathogenicity related to a certain extent. All of these methods provide some information on pathogenicity, but no single method seems to be universally valid.

The aim of our study was to correlate various qualities of Acanthamoeba strains and to evaluate any consistencies among clinically relevant isolates and among isolates of no clinical relevance. Isolates were identified by morphological and molecular biological means. Cluster analysis based on 18S rDNA sequences was performed, and the isolates were grouped according to their pathogenicity-related properties.

	MATERIALS AND METHODS

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Amoebae. A total of 13 Acanthamoeba strains of various clinical relevance, including 1 strain from the brain of a mouse, 1 strain from the necrotic tissue of a basilisk, 4 strains from Acanthamoeba keratitis patients, 3 strains from non-Acanthamoeba keratitis patients, 2 strains from contact lens cases of asymptomatic individuals, 1 strain from the nasal mucosa of an asymptomatic individual, and 1 strain from tap water, were investigated and compared (Table 1).

Isolation. The amoebae were isolated from clinical specimens by the plate culture method. Corneal epithelium and swabs from contact lens cases were inoculated onto non-nutrient agar plates seeded with a 48-h-old culture of Escherichia coli. Initial cultures were diluted in order to eliminate cocontaminants by cutting a small piece of agar with a sterile scalpel and applying it centrally onto a fresh plate. All isolates were cloned by transferring a single cyst to a fresh plate employing a micromanipulator.

Morphological classification. Morphological studies included measurement of trophozoites and cysts and assessment of the size and shape of the endo- and ectocysts and of the mean number of opercula and resulted in the classification of the isolates as Acanthamoeba sp. groups I, II, or III. Species identification was achieved according to the identification key of Page (20) based mainly on cyst morphology and temperature tolerance.

Isolation of DNA. Whole-cell DNA was isolated by a modified UNSET (14) procedure. In brief, amoebae (~10⁶ cells) were harvested from extensively growing axenic cultures by centrifugation at 500 × g for 7 min. From strains which would not grow in axenic culture, DNA was isolated directly from the plate culture as described previously (29). The amoeba pellet was resuspended in 500 µl of UNSET lysis buffer, overlaid with 500 µl of phenol-chloroform-isoamylalcohol (PCI), and shaken gently for 5 h. The suspension was centrifuged at 3,000 × g for 10 min, and the upper, aqueous phase was transferred to a new tube. PCI extraction was repeated two times for 10 min each time. Nucleic acids were precipitated by ethanol (15 min at 80°C), pelleted at 12,000 × g for 30 min at 4°C, washed in 70% ethanol, air dried, and resuspended in 30 µl of sterile double-distilled water. The 18S rRNA gene was amplified using the SSU1 and SSU2 primers (9), which are complementary to the 5' and the 3' end of the gene, respectively. We used 0.8 µl of whole-cell DNA and a standard amplification program (30 cycles of 95°C for 1 min, 50°C for 2 min, and 72°C for 3 min). Amplification of the 18S rRNA gene was visualized by ethidium bromide staining in agarose gel electrophoresis. The amplified gene was sequenced stepwise by direct sequencing from the PCR product using the Thermo Sequenase II sequencing kit (Amersham Pharmacia Biotech GmbH, Vienna, Austria) and the subsequent construction of complementary internal primers. Sequences were obtained from both strands. Sequencing was carried out in a 310 ABI PRISM automated sequencer (PE Applied Biosystems, Langen, Germany), and sequence data were processed with the GeneDoc (18) sequence editor.

Identification of sequence types and dissimilarity calculation. The sequence types for the 312-2, 36KL, 7AR, 1BU, 2HH, 3ST, 4CL, and 5SU strains were determined by searching for the published sequence with the greatest nucleotide identity employing the BLAST application (1) and by subsequently calculating the percentage of nucleotide dissimilarity using all sites of the gene. Stothard et al. (26) have established their sequence types with <4.9% nucleotide dissimilarity within the sequence types and >5% nucleotide dissimilarity between different sequence types. Multiple sequence alignment was performed by subsequent pairwise alignment using the CLUSTAL X application (27) and showed homologous positions. A dissimilarity matrix was calculated using all sites of the gene.

Cluster analysis. Cluster analysis was performed for the T4 isolates by constructing a cladogram using the PHYLIP (7) package. The tree was rooted by sequence types T3 and T11 using the T3 strains S-7 (A. griffini; GenBank accession no. U07412), Hay & Seal (A. griffini; S81337), Panola Mt. (A. polyphaga; AF019052), and Sawyer (A. pearcei; AF019053) and the T11 strains RB:F:1 (A. stevensoni; AF019069), BH-2 (A. hatchetti; AF019068), and our T11 isolate 4RE (A. hatchetti; AF251937) as an outgroup.

Growth rates. Since several strains did not grow in axenic culture, growth rates were evaluated on a lawn of living E. coli in the plate culture. Amoeba cysts were harvested from plate cultures, counted in a hemacytometer, and brought to a concentration of 10⁵ cells/ml. Then, 1 µl was inoculated into the center of an agar plate seeded with 100 µl of a 48-h-old culture of E. coli. The cultures were incubated at a range of temperatures (30, 34, 37, 40, and 42°C) and observed daily using phase-contrast microscopy. In the presence of bacteria the amoebae excyst and, feeding on the bacteria, constantly proliferate by binary fission. Replacing the bacteria, the amoeba front moves toward the edges of the plate. Plates were marked with three concentric circles spaced at 15-mm intervals on the bottom side. Growth rates were recorded after 48 h and assessed according to the number of circles already traversed by the amoeba front. The ability of the amoebae to migrate into the agar was recorded after 72 h, and the temperature tolerances were recorded after 2 weeks.

Cytopathic effects. HEp-2 cells were cultured in a 1:1 mixture of PC-1 (BioWhittaker, Walkersville, Md.) and CO₂-independent medium (Life Technologies, Ltd., Paisley, Scotland) supplemented with L-glutamine (2 mM) in 75-cm² tissue culture flasks (Corning/Costar, Bodenheim, Germany) at 37°C under sterile conditions. Then, 1 ml of a 10⁵-cell/ml axenized suspension of each isolate was inoculated onto a monolayer of HEp-2 cells. Cocultures of amoebae and tissue cells were incubated at 30, 37, and 42°C. Pathogenicity was defined as complete lysis of the monolayer within 48 h at 30°C. All experiments were carried out in triplicate and were repeated after 6 weeks.

Sequence data. Sequence data were deposited in GenBank and are available under the following accession numbers: AF260718 (strain 312-2), AF260719 (strain 36KL), AF260720 (strain 7AR), AF260721 (strain 1BU), AF260722 (strain 2HH), AF260723 (strain 3ST), AF260724 (strain 4CL), and AF260725 (strain 5SU).

	RESULTS

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Identification of strains. The five new isolates (1BU, 2HH, 3ST, 4CL, and 5SU) all exhibited a typical Acanthamoeba group II morphology. 2HH and 3ST were assigned to A. hatchetti. The 1BU and the 4CL strains were designated to A. castellanii, and the 5SU strain was identified as A. polyphaga. Altogether, the species spectrum in our study comprised four strains of A. hatchetti, four strains of A. castellanii, and one strain each of A. lenticulata, A. palestinensis, A. polyphaga, A. quina-lugdunensis, and A. rhysodes.

Cluster analysis. In order to evaluate whether this grouping is also valid in a phylogenetic context, the T4 isolates were grouped according to their 18S rDNA sequences by constructing a cladogram (Fig. 1). T3 and T11 strains were used as an outgroup since these are the closest related sequence types to type T4 (26). Our analysis revealed high bootstrap values and also, if maximum parsimony or other outgroups were used, the clustering of strains remained in the same order. Cluster analysis revealed that all T4 isolates formed a monophyletic group. Interestingly, within type T4 the strains of no clinical relevance (36KL, 5SU, 9GU, 4CL, 7AR, and 312-2) clustered together (Fig. 1).

View larger version (11K): [in this window] [in a new window]   FIG. 1.   18S rDNA neighbor-joining tree of the 10 sequence type T4 isolates. The tree was rooted by T3 and T11 using the T3 strains S-7, Hay & Seal, Panola Mt., and Sawyer and the T11 strains RB:F:1, BH-2 and the T11 isolate 4RE as an outgroup. Bootstrap values are based on 1,000 replicates. The numbers at the nodes represent bootstrap values based on 1,000 replicates.

Physiological characterization. Generally, isolates that clustered together in the sequence analysis were rather similar in their physiological properties, and moreover, physiological characteristics of the isolates corresponded very well to their initial clinical relevance values (Table 3). While virulent strains exhibited high growth rates, high temperature tolerances, and high cytopathic effects, the strains isolated from contact lens cases or sanitary facilities grew only moderately, preferentially at lower temperatures, and showed low cytopathic effects. All strains except the 36KL isolate showed growth at 37°C, and eight strains showed the ability to grow at 40°C. However, all five strains growing at 42°C were among the clinical isolates. The physiological characteristics of the 40AB strain with previously unknown clinical relevance suggest that this strain is virulent.

	DISCUSSION

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Identification. Apart from the reference strain 72/2 and the 36KL strain, all investigated isolates displayed an Acanthamoeba group II morphology. Also, in a previous study of 18 cases of Acanthamoeba-associated keratitis in Austria, all Acanthamoeba isolates exhibited a group II morphology (Walochnik et al., submitted). In fact, group II seems to be the most abundant group not only in the environment but also in clinical specimens (26).

Pathogenicity. Stothard et al. (26) reported on 29 of 30 analyzed Acanthamoeba keratitis causing strains belonging to sequence type T4. They anticipate that the ability to cause keratitis might have only evolved once, since the only exception in their study is a T3 strain and this sequence type is, other than type T11, the sequence type most closely related to sequence type T4.

Cluster analysis. This grouping was also confirmed by cluster analysis, within type T4, isolates with no clinical relevance clustered together. It has been reported that restriction endonuclease digestions of Acanthamoeba whole-cell DNA reveal a distinct RFLP type being most frequently associated with keratitis (16), and comparison of mitochondrial DNA digests also indicate the presence of coherent cladistic populations among the clinical Acanthamoeba isolates (30). In a recent study on mitochondrial DNA digestion patterns, it was shown that 80% of the human isolates studied group into 7 of 22 genotypes, whereas the other 20% were distributed among the 15 other genotypes; the authors assume that virulence may be associated with specific clusters of cladistic groups within Acanthamoeba (31). Also, in Acanthamoeba group III, a grouping according to pathogenicity has been observed with A. griffini branching between pathogenic and nonpathogenic isolates (32). The recent finding of an eye and a lung isolate being most closely related with regard to their 18S rDNA sequences confirms this assumption. The authors concluded that any pathogenic isolate may be capable of infecting more than one tissue (9). It seems at least conceivable to us that any isolate infecting the brain can also infect the eye, an immunologically underprivileged site.

Physiological properties. Remarkably, sequence similarity clustering was strongly supported by the pathogenicity-associated physiological properties of these isolates. Altogether, clinically relevant strains grew significantly better than strains isolated from non-Acanthamoeba keratitis patients. Yagita et al. assume that the development of an eye infection may depend on the size of the inoculum, the frequency of contact with the cornea, and the virulence of the amoeba (31). In this respect the growth rate is of prime importance in order to provide a large inoculum. There was a true correlation of growth rate and cytopathic effect in our study. Moreover, the 42°C growing strains were all among the keratitis-causing isolates, although temperature tolerance is probably less important for infections of the eye since the human eye has an average temperature of only 34°C. We also observed differences in the ability of isolates to migrate into the agar. It is assumed that the amoeboidal locomotion enables the amoebae to penetrate host tissue. In Naegleria spp., migration patterns in an under-agarose system correlate with pathogenicity (28).