PREFERENTIAL GROWTH TEMPERATURES OF T4 AND T5 THAI SOIL
Acanthamoeba ISOLATES
Atsadang Boonmee1,3*, Saradee Warit2, Araya Chusattayanond3,#
1
Master of Science Program in Microbiology (International program), Department of
Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
2
TB Research Laboratory, National Center for Genetic Engineering and Biotechnology,
NSTDA, Pathumthani 12120, Thailand
3
Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400,
Thailand
*e-mail: atsadang88@gmail.com #e-mail: arayachusattayanond@gmail.com
Abstract
Acanthamoeba, a genus of free-living protozoa, can cause a sight-threatening
Acanthamoeba keratitis (AK) following corneal trauma or inappropriate use of contact lenses.
Sinusitis, skin ulcer and granulomatous amoebic encephalitis (GAE) can occur in
immunocompromised and some normal individuals. Acanthamoeba is classified into 17
genotypes (T1-T17) based on nucleotide sequences of 18S rRNA gene. Members of T4 and
T5 are the most and second most abundant populations in environments. Surprisingly, while
the majority of pathogenic Acanthamoeba belong to T4, only 2 T5 infected AK and 1 GAE
cases were reported worldwide. Understanding the biological differences among
Acanthamoeba genotypes may help understanding their epidemiology, disease prevention,
pathogenesis and treatment against their infections. In this study, 20 soil Acanthamoeba
isolates identified as 13 T4 and 7 T5 genotypes, according to nucleotide sequences of
GTSA.B1 region in 18S rRNA gene, were studied for preferential growth temperatures under
30°C, 34°C, 37°C, and 42°C. It is obvious that all isolates can grow at 30-37°C, but only T5
isolates can grow well at 42°C. Preferential growth temperatures were found to vary among
different isolates. It seems not to be genotype dependent. Most of T4 (53.85%) and T5
(42.86%) isolates seem to prefer growing at 34°C to other test temperatures. However, only
Aud8, S3, SamP1, and Udon1 showed statistically significant level of the preference.
Keywords: soil Acanthamoeba, GTSA.B1, T4, T5, growth temperature
Introduction
Acanthamoeba is a genus of free-living protozoa. The organism is widely distributed
in environments such as soil, fresh water, tap water, ocean, and air-conditioning units. There
are two stages in its life cycle. One is the feeding and reproducing trophozoite. The other one
is the resistant cyst which is formed mainly under hash condition such as depleted food
storage, extreme temperatures and pH. Cyst is enclosed in double walls consisted of the outer
ectocyst and the inner endocyst (1). They can cause Acanthamoeba keratitis (AK),
granulomatous amoebic encephalitis (GAE), nasopharyngeal and cutaneous lesions (2). AK
can be found following inappropriate use of contact lenses or corneal trauma, while GAE is
an opportunistic and fatal disease occurred in immunocompromised individuals. However, a
few GAE cases of immunocompetent persons have also been reported (3).
Acanthamoeba have previously been classified into three groups based on their
morphology. They include astronyxid, polyphagid, and culbertsonid (4). However,
polymorphism of cysts within a cloned cell line was reported when different media and
culture conditions were used. The inability to differentiate cyst morphology accurately has
made the morphological taxonomic scheme unreliable and difficult (5). At present, nucleotide
sequencing of 18S rRNA gene is the most reliable method to differentiate among
Acanthamoeba members. This is due to the appropriately large number of variable bases in
this gene and the availability of a large number of known sequences in the data base (6).
Schroeder (6) reported that 1500 bp GTSA.B1 region of 18S rRNA gene is appropriate for
Acanthamoeba genotyping. This Acanthamoeba genotype-specific amplimer contains 8
variable regions sufficient to differentiate among Acanthamoeba genotypes. (6). Up to date,
17 genotypes including T1-T12, (7, 8), T13 (9), T14 (10), T15(11), T16 (12), T17 (13) have
been discovered with a divergence of more than 5% from the most closely related genotypes.
T4 is the most abundant genotype consisted of the largest Acanthamoeba population. Up to
90% of the reported T4 isolated were obtained from AK patients (6, 8). It is interesting that
T5 is the second most abundant population, but only two cases of AK and one GAE patients
have been reported (14-16).
Shoff et al. (17) reported that T3, T5 and T11 Acanthamoeba isolated from
environments showed a significantly higher degree of resistance to disinfectants than T4
members isolated from corneal scrapings and beaches. In addition, T3, T5 and T11
Acanthamoeba isolates also showed the ability to grow at temperatures as high as 41°C.
These findings suggested that Acanthamoeba belonging to different genotypes have different
degree of temperature tolerance. On the other hand, it may suggest that preferential growth
temperatures of Acanthamoeba belonging to different genotypes are also different.
While Acanthamoeba spp. are ubiquitously found in environments and can cause
sight- and life threatening diseases, knowledge about their biological properties and several
physical characteristics related to their pathogenicity and drug susceptibility are still limited.
We believe that understanding about Acanthamoeba biological and physical characteristics
will enable the development of useful tools for disease prevention, therapy and elimination or
controlling their population in selected areas. Therefore, this study is aimed at determining
the preferential growth temperatures of soil Acanthamoeba belonging to T4 and T5
genotypes. Thirteen isolates of T4 and seven isolates of T5 Acanthamoeba identified by
sequencing of GTSA.B1 region in 18s rRNA gene were studied under 30C to 42C.
Methodology
Acanthamoeba spp.
Twenty isolates of Acanthamoeba spp. were obtained from soil samples randomly
collected from 14 provinces of Thailand including Aud4, Aud8, Aud9 from Uttradit, Ayut2,
Ayut3, Ayut6 from Phra Nakhon Si Ayutthaya, Chon3, Chon9 from Chon Buri, KK1 from
Khon Kaen, Nas5 from Nakhon Sawan, Pat9 from Phatthalung, Rach6 from Nakhon
Ratchasima, Roi5 from Roi Et, S3 from Bangkok, SamP1 from Samut Prakan, SamSa3 from
Samut Sakhon, Sri1, Sri6 from Si Sa Ket, Ubol6 from Ubon Ratchathani and Udon1 from
Udon Thani. They were cultured and maintained monoxinically on 2% non-nutrient agar
using heat-killed E. coli as nutrient source. From each of the isolates, a single cyst was
carefully picked up and transferred to a fresh monoxenic culture plate. Clones of the singly
isolated acanthamoebae were established and maintained at room temperature.
Acanthamoeba genotyping
Approximately one million trophozoites were prepared for DNA extraction.
Trophozoites were pelleted down at 1,500 rpm, 20°C for 5 min. Trophozoites were lysed by
resuspending in 100 μl of lysis buffer (50 mM NaCl, 10 mM EDTA, 50 mM Tris-Cl pH 8.0)
and 2.5 μl of proteinase K as previously described by Sambrook et al., 1989 (17). The DNA
extraction was performed by using TRIZOL/chloroform as recommended by the
manufacturer (Invitrogen). Acanthamoeba genotype was identified according to the
sequences of their GTSA.B1 region in 18S rRNA gene as previously described by Schroeder
et al. (6). GTSA.B1 sequences were amplified by CRN5 and 1137 primers using GoTag®
Green Master Mix (Promega) as recommended by the manufacturer. PCR cycles were
programmed to run a cycle of 7 min at 95°C, followed by 20 cycles of 1 min at 95°C, 1 min
at 60°C, and 2 min at 72°C and finished with 25 cycles of 1 min at 95°C and 2 min at 72°C
(6). The PCR products were sent out to Macrogen, Korea for purification and CRN5, 373,
570C, 892, 892C and 1137 primers were used for bidirectional sequencing. The obtained
sequences were aligned and edited by BioEdit (version 7.1.3.0). NCBI BLAST was used to
search for similar sequences in the GenBank.
Determination of preferential growth temperatures
Preferential growth temperatures of 20 Acanthamoeba isolates were determined by
the technique modified from those previously described (18, 19). In this study, three
concentric circles spaced by 15 mm intervals were drawn with a water-insoluble marker on
the bottom of each monoxenic culture plate. A drop of 105 Acanthamoeba cysts in 5 μl
amoeba saline (KCl: 0.4g, CaCl2: 3g, MgSO4.7H2O: 1g, distilled water to 1000 ml) was
inoculated onto the centre of a culture plate and left for 72 h. Growth of each Acanthamoeba
isolate was assayed under four different temperatures including 30°C, 34°C, 37°C and 42°C.
After 72 h of incubation, number of trophozoites and cysts in the colony spreading over the
marked circles were manually counted under an inverted light microscope.
Statistical analysis
The numbers of Acanthamoeba (trophozoites + cysts) obtained from each isolate at
different temperatures were compared statistically using one way ANOVA. The analysis was
implemented using Prism 5 version 5.00. p-value lower than 0.05 was considered statistically
significant.
Results
Acanthamoeba spp. genotyping
Twenty Acanthamoeba isolates were blasted and the results showed 13 isolates with
high score of identity (97-100%) to Acanthamoeba belonging to T4 genotype. Seven other
isolates showed the maximum identity (97-100%) to Acanthamoeba belonging to T5
genotype. The accession numbers of the top hit score were shown in Table1.
Table 1. BLAST results showing query cover, maximum identity percent, accession numbers and
genotypes of the top hit results of all 20 Acanthamoeba isolates
Isolates
Aud4
Aud8
Aud9
Ayut2
Ayut3
Ayut6
Chon3
Chon9
KK1
Nas5
Pat9
Rach6
Roi5
S3
SamP1
SamSa3
Sri1
Sri6
Ubol6
Udon1
Description
Acanthamoeba sp. Ac_E11
Acanthamoeba sp. Ac_E3
Acanthamoeba polyphaga ATCC 50371
Acanthamoeba sp. Ac_E11
Acanthamoeba sp. Ac_E8a
Acanthamoeba lenticulata strain PD2S
Acanthamoeba sp. ATCC 50369
Acanthamoeba sp. ATCC 50369
Acanthamoeba lenticulata strain PD2S
Acanthamoeba lenticulata strain 68-2
Acanthamoeba sp. Ac_E11
Acanthamoeba lenticulata strain PD2S
Acanthamoeba sp. Ac_E11
Acanthamoeba sp. Ac_E5a
Acanthamoeba lenticulata strain PD2S
Acanthamoeba sp. Ac_E8c
Acanthamoeba sp. ATCC 50369
Acanthamoeba lenticulata strain PD2S
Acanthamoeba lenticulata strain 72/2
Acanthamoeba sp. ATCC 50369
Query
cover
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
99%
100%
100%
100%
100%
100%
100%
100%
100%
100%
Ident
99%
99%
97%
100%
99%
99%
100%
99%
100%
100%
99%
100%
99%
99%
99%
99%
100%
99%
100%
100%
Accession
numbers
GU808306
GU808283
U07407
GU808306
GU808298
U94741
U07409
U07409
U94741
U94733
GU808306
U94741
GU808306
GU808289
U94741
GU808300
U07409
U94741
U94732
U07409
Genotype
T4
T4
T4
T4
T4
T5
T4
T4
T5
T5
T4
T5
T4
T4
T5
T4
T4
T5
T5
T4
The sequences of 20 isolates were submitted to GenBank. Accession numbers are
shown as follow: Aud4 (KF733228), Aud8 (KF733231), Aud9 (KF733232), Ayut2
(KF733225), Ayut3 (KF733226), Ayut6 (KF733227), Chon3 (KF733235), Chon9
(KF733237), KK1 (KF733238), Nas5 (KF733240), Pat9 (KF733243), Rach6 (KF733245),
Roi5 (KF733249), S3 (JX104341), SamP1(KF733254), SamSa3 (KF733256), Sri1
(KF733257), Sri6 (KF733258), Ubol6 (KF733261), and Udon1 (KF733263).
Preferential growth temperatures
Different isolates of the T4 Acanthamoeba tested in this study showed different
preferential growth temperatures. They can be grouped as follow. Aud4, Aud9, Pat9, and Sri1
prefer to grow at 30°C. However, only the growth of Pat9 at this temperature is statistically
significant different (p≤0.05) from the growth at other temperatures (see Fig. 1). Another
group of seven isolates: Aud8, Ayut2, Ayut3, Chon9, S3, SamSa3, and Udon1 prefer to grow
at 34°C, but only Aud8, S3 and Udon1 showed statistical significant number (p≤0.05) of the
grown up population (see Fig. 2). On the other hand, Chon3 and Roi5 isolates prefer to grow
under a slightly higher temperature (37°C) (see Fig. 3). Most of the isolates did not grow at
42°C, but a few trophozoites of Pat9 (5 cells), Roi5 (8 cells), and Udon1 (1 cell) were found
on the culture plates. It can be concluded that 7 of 13 T4 isolates (53.85%) prefer to grow
under 34°C.
Figure 1. A histogram showing the numbers of Aud4, Aud9, Pat9 and Sri1 T4 Acanthamoeba isolate
grown at 30°C, 34°C, 37°C and 42°C for 72 h. Each number was obtained as a mean value of a triplicate
experiment. * P ≤ 0.05 was obtained by one-way ANOVA comparing the cell numbers at different
temperatures.
Figure 2. A histogram showing the numbers of Aud8, Ayut2, Ayut3, Chon9, S3, SamSa3, and Udon1 T4
Acanthamoeba isolates grown at 30°C, 34°C, 37°C and 42°C for 72 h. Each number was obtained as a
mean value of a triplicate experiment. * P ≤ 0.05 were obtained by one-way ANOVA comparing the cell
numbers at different temperatures.
Figure 3. A histogram showing the numbers of Chon3 and Roi5 T4 Acanthamoeba isolates grown at 30°C,
34°C, 37°C and 42°C for 72 h. Each number was obtained as a mean value of a triplicate experiment. The
cell number differences are not statistically significant (P > 0.05) according to one-way ANOVA.
Unlike the T4 isolates, all 7 T5 Acanthamoeba isolates survived and grew under
42°C. This might be a specific characteristic of T5 Acanthamoeba genotype. On the other
hand, preferential growth temperatures of these T5 members were found to differ among
different isolates. As shown in Fig. 4, the preferential temperature of Sri6 is 30°C (p≤0.05),
while Ayut6, SamP1 and Ubol6 seem to prefer 34°C to other test temperatures. However,
only SamP1 showed significant level (p≤0.05) of the preference at 34°C (see Fig. 5). On the
other hand, KK1 and Rach6 prefer a slightly higher temperature at 37°C (see Fig. 6), while
Nas5 prefers high temperature at both 37°C and 42°C (see Fig. 7). Nevertheless, it is obvious
that the majority of T5 population in this study grew well under 34°C, the temperature that
T4 isolates can also grow well.
Figure 4. A histogram showing the numbers of Sri6 T5 Acanthamoeba isolate grown at 30°C, 34°C, 37°C
and 42°C for 72 h. Each number was obtained as a mean value of a triplicate experiment. * P ≤ 0.05 was
obtained by one-way ANOVA comparing the cell numbers at different temperatures.
Figure 5. A histogram showing the numbers of Ayu6, SamP1, and Ubol6 T5 Acanthamoeba isolates grown
at 30°C, 34°C, 37°C and 42°C for 72 h. Each number was obtained as a mean value of a triplicate
experiment. * P ≤ 0.05 was obtained by one-way ANOVA comparing the cell numbers at different
temperatures.
Figure 6. A histogram showing the numbers of KK1 and Rach6 T5 Acanthamoeba isolates grown at 30°C,
34°C, 37°C and 42°C for 72 h. Each number was obtained as a mean value of a triplicate experiment. The
differences are not statistically significant different (p > 0.05) according to one-way ANOVA comparisons.
Figure 7. A histogram showing the numbers of Nas5 T5 Acanthamoeba isolate grown at 30°C, 34°C, 37°C
and 42°C for 72 h. Each number was obtained as a mean value of a triplicate experiment. The cell numbers
among different time points are not statistically significant different (P > 0.05) according to one-way
ANOVA.
Discussion and Conclusion
In this study our 13 Acanthamoeba isolates were classified as the members of T4
Acanthamoeba genotype, and 7 other isolates were the members of T5. This result is
consistent with those previously reported that Acanthamoeba belonging to T4 and T5
genotypes are the most and the second most abundant populations distributed world-wide
(16, 20). According to the finding of Booton et al. (16), it is presumed that the majority of
AK isolates (94%) belonged to T4 genotype because of the existence of the largest population
of T4 isolates in the environments. This might be due to the following possibilities. 1) T4
Acanthamoeba have adapted themselves to various conditions of the environments. 2) They
have developed the ability to reproduce numerously. 3) They have developed the ability to
resist the contact lens cleaning solutions.
According to our results, all Acanthamoeba isolates in this study can grow under 3037°C. Most of those belonging to T4 genotype did not grow well at high temperature (42°C),
while the T5 members can grow well at this high temperature. This ability to grow under high
temperature may be due to the presence of specific gene evolved along its evolution. Thus the
capability to survive at high temperature is one of the biological characteristics of
Acanthamoeba belonging to T5 genotype. Therefore, cultivation at 42°C may be used as a
selective culture condition for obtaining T5 Acanthamoeba. It is quite interesting that T5
Acanthamoeba were previously found to be resistant to fusaric acid, a proven
acanthamoebicidal agent obtained from endophytic fungus Fusarium sp. Tlau3 (22), and
multipurpose contact lens cleaning solutions (21).
From the present study, we also found that T5 Acanthamoeba differs morphologically
from the members of T4. T5 cysts are clearly recognized as global- double-walled shape,
while cysts of T4 can be star-like or polygonal shaped. This finding corresponds well with the
available information previously described (1). The unique characteristics of T5
Acanthamoeba found in this study together with those previously reported may suggest that
T5 may overgrow and predominate under the global warming. Treatment against T5 infection
seems to be more problematic according to the finding that T5 is more resistant to
acanthamoebicidal agent (22) and contact lens cleaning solution (21).
The ability of T4 and T5 to grow at 34°C is another interesting point since it is the
temperature of human eyes (19). This finding corresponds well with the fact that most of
Acanthamoeba infections involve the cornea. The ability of some isolates to grow quite well
at 37°C may explain the occurrence of disseminated acanthamoebiasis.
In this study, we cannot specify the preferential growth temperatures of
Acanthamoeba belonging to genotype T4 and T5 because the preferential growth temperature
varies among isolates of different genotypes.
Further study using more genotypes and more isolates will provide more
understanding about biological and physical characteristics of Acanthamoeba spp.
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Acknowledgements
This research was supported by Science Achievement Scholarship of Thailand from
the Higher Education, Ministry of Education, Thailand.