Vol.3, No.8, 529-533 (2011) Health
Copyright © 2011 SciRes. Openly accessible at http:// www.scirp.org/journal/HEALTH/
ITS and pB2.5 gene expression of Naegleria fowleri in
drug resistance
Jundee Rabablert1, Supathra Tiewcharoen2*, Virach Junnu2
1Department of Biology, Faculty of Science, Silpakorn University, Nakhon Pathom, Thailand;
2Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand;
*Corresponding Author: sistc@mahidol.ac.th
Received 8 May 2011; revised 22 June 2011; accepted 13 July 2011.
Naegleria fowleri was causative agent of pri-
mary amoebic meningoencephalitis (PAM). Ac-
croding to the failure of treatment, several re-
searches reported the activity of chemothera-
peutic drugs against N. fowleri but we did not
know the drug resistance of the amoebae. The
purpose of this study was to examine the ef-
fects of drugs (amphotericin B, artesunate, azi-
thromycin, voriconazole, chlorpromazine, fluco-
nazole and gentamicin sulphate) on ITS and
pB2.3 genes of Naegleria fowleri trophozoites.
Our study demonstrated gene expression of
treated N. fowleri by RT-PCR. The results re-
viewed that ITS gene of N. fowleri showed up
regulate to amphotericin B, azithromycin and
gentamicin sulphate, while pB2.3 gene showed
up regulate to artesunate. These results com-
pared with beta actin (house keeping gene) ex-
pression at time intervals 15 - 120 min. The cha-
nge of gene expression of treated N.fowleri was
possibly to cause of drug resistance. The me-
chanism of drug resistance genes ITS and pB2.3
of N. fowleri should be cl arified in further study.
Keywords: Naegleria Fowleri; ITS; pB2.3; Drug
Naegleria fowleri causes severe meningoencephalitis
mainly in children and young adults. Due to the treat-
ments have not been succeeded, most of patients die
from N. fowleri infection [1]. The effect of drug against
N. fowleri has been carried out both in vitro and in vivo
studies which provided for clinical trends for treatment
[2]. A prelude of drug resistant has been frequently
documented in recent years [3]. Several researches on
antifungal resistant have been focused on elucidating the
molecular basis and transcriptional regulation of azole in
Candida spp. [4]. The resistance to azole uptake of C.
albicans can be achieved with the introduction of key
point mutation in and/or up regulation of gene expres-
sion which encodes on efflux pump; EGR11, MDR in-
cluding ATP-binding cassette transporter molecules CDR2
[5]. In addition, antifungal resistant was involved sterol
uptake which was controled by UPC2 gene [6]. Owing
to failure treatment of PAM, one of the major problems
was drug resistant from gene alteration. Up to date, a
study has been addressed the intrinsic resistant on nfa1
and Mp2Cl5 genes which regulated pathogenesis of the
amoebae. The results demonstrated that either nfa1 re-
sistant to fluconazole or Mp2Cl5 resistant to amphote-
ricin B, azithromycin and artesunate of N. fowleri were
found [7]. Owing to the dominant ITS, located in the
5.8S rRNA gene and species-specific chromosomal DNA
pB2.3 genes were used for identify pathogenic N. fowleri
[8] and diversity of N. fowleri at molecular level [9], we
investigated the activity of drugs on ITS and pB2.3 genes
of N. fowleri. This report revealed the ability of Naegle-
ria genes against drugs of choice.
2.1. Naegleria Fowleri Cultivation
Free living N. fowleri trophozoites (Khon-Kaen strain)
were cultured in Nelson’s medium supplemented with
5% heat-inactivated fetal calf serum (FCS) without anti-
biotics at 37˚C. Trophozoites were tested with the con-
centration of amphortericin B, voriconazole, fluconazole,
chlorpromazine, artesunate, azithromycin and gentami-
cin at IC 50 [10] during 15 - 120 min, triplicate. Untreated
trophozoite was used for negative control. At indicated
times, trophozoites were twice washed with normal sa-
line and frozen at –80˚C until required.
2.2. RNA Extraction
Total RNA was extracted from untreated or treated
J. Rabablert et al. / Health 3 (2011) 529-533
Copyright © 2011 SciRes. Openly accessible at http:// www.scirp.org/journal/HEALTH/
amoebae trophozoites using Tri Reagent (Sigma-Aldrich,
USA). For positive control, nf actin gene (housekeeping
gene) of N.fowleri was confirmed by primer 5′́̀- ACT
TGA CAA TTT CTC TCT CAG TGG-3. The amplicons
of amoebae were prepared from primers of ITS (ITS1;
TTTCTTTTCCTCCC CTTATTA-3) and pB2.3 (p3f; 5-
Super-script PCR (Invitrogen, Gransland). MW of each
amplicon was detected by 1.5% Agarose Gel Electro-
phoresis at 100 V for 30 min.
In our studies we demonstrated the responsibility of N.
fowleri to the effects of drugs (amphotericin B, artesu-
nate, azithromycin, voriconazole, chlorpromazine, flu-
conazole and gentamicin sulphate) on ITS and of pB2.3
gene by RT-PCR. The number of N. fowleri treated or
untreated N. fowleri was not significant different be-
tween two groups at time intervals (the results was not
shown) (t < 0.005). Total RNA was extracted from this
two groups using Tri Reagent (Sigma-Aldrich, USA) at
indicated time. As a positive control, we used nf actin to
amplify under the same RT-PCR condition and regula-
tion of expression of nf actin gene was detected at 170
bp at 15 - 120 min. The up regulate of nf actin was com-
pared with treated or untreated amoebae at every point
of time. We found that untreated N.fowleri showed up
regulate of ITS gene at 450 while ITS gene expression
from treated trophozoites with voriconazole, fluconazole
or chlorpromazine was not observed during 120 min. In
contrast, trophozoites treated with amphotericin B was
found at least 30 min whereas trophozoites treated with
azithromycin or gentamicin was shown at least 45 min
(Figure 1). Similarly, we tested the drugs activity against
pB2.3 gene expression of amoebae trophozoites. The
untreated trophozoites showed bright band fragment at
310 bp as shown in Figure 2. Interesting, we did not
observe pB2.3 gene expression from treated trophozoites
with a panel of drugs at 120 min, except artesunate. It is
possibly suggested that ITS gene of N. fowleri tropho-
zoite was resisted to amphotericin B, azithromycin or
gentamicin including the pB2.3 gene was resisted to ar-
Figure 1. Effect of drugs on ITS gene of N. fowleri by RT-PCR at 15, 30, 45, 60 and 120 min (a1-e1)
compared with the positive expression control, nf actin gene at the same time (a2-e2). Untreated N.
fowleri showed bright band fragment at 450 bp (lane1). Treated N. fowler i with amphotericin B (lane
2), voriconazole (lane 3), fluconazole (lane 4), chlorpromazine (lane 5), artesunate (lane 6), azithro-
mycin (lane 7), and gentamicin sulphate (lane 8), respectively.
J. Rabablert et al. / Health 3 (2011) 529-533
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Figure 2. Effect of drugs on pB2.3 gene of N. fowleri by RT-PCR at 15, 30, 45, 60 and 120 min (f1-j1) com-
pared with the positive expression control, nf actin gene(170) at the same time (f2-j2). Untreated N. fowleri
showed bright band fragment at 310 bp (lane1). Treated N. fowleri with amphotericin B (lane 2), voriconazole
(lane 3), fluconazole (lane 4), chlorpromazine (lane 5), artesunate (lane 6), azithromycin (lane 7), and gen-
tamicin sulphate (lane 8), respectively.
Amphotericin B has been generally recognized for
fungal and protozoa treatment, many publications re-
ported amphotericin B-resistant genes in Candida lusita-
niae, Saccharomyces cerevisiae [11] and Cryptococcus
neoformans [12]. However, it has been reported in path-
ogenic protozoa; Entamoeba histolytica, Giardia lam-
blia, Trichomonas vaginalis and also occurred anaerobic
protozoa; Blastocystis hominis, Cry p tospo r idium p arvum,
Isospora spp., Cyclosporssporidia spp. [13]. In addition,
amphotericin B resistant gene was also found in vector
born protozoa, Leishmania tarentolae [14].
The mechanism of amphotericin B, polyene resistant
of C. albicans and S. cerevisiae caused by mutation of
EGR genes which control the production of ergosterol
and sensitivity to polyenes. As the result of EGR6 mu-
tant strain of C. lusitaniae transcription, the reduced
ergosterol content was appeared [15]. A study of EGR6
mutant caused unable to form amphotericin B-generated
pores in the cell membrane on Candida spp [16]. Our
study revealed amphotericin B resistant ITS gene of N.
fowleri was firstly appeared. Moreover, drug resistant;
azithromycin, gentamicin sulphate to ITS and artesunate
to pB2.3 of N. fowleri were also demonstrated. A few
publications reported the azithromycin resistant in Pseu-
domonas aeruginosa [17] and genetic mutation at 23S
rRNA region of Ureaplasma urealyticum and Neisseria
gonorrhoeae [18]. Gene resistant, ermG , to azithromy-
cin was established in Bacteroides. Mechanism of azith-
romycin resistant established at MexCD-OprJ pump of
Pseudomonas aeruginosa biofims [19].
Gentamicin has been general used in cultivation; En-
terococcus faecali [20], Pseudomonas aeruginosa [21].
The occurrence of gentamicin-resistant genes of gram
negative bacteria; Enterobacteriaceae, Pseudomonas,
Acinetobacter were isoloated from different environment
mainly originating from sewage, faces, coastal water
J. Rabablert et al. / Health 3 (2011) 529-533
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polluted with wastewater and the distribution of these
resistant genes could broadly transfer to hosts [22].
Gentamicin-resistant to N. fowleri has been found in
vitro since 2009 [10].
This study showed that gentamicin resistant concerned
both gene expression and aminoglycoside activity [20].
Artesunate was used to treatment for falciparum malaria
due to artesunate accumulated lipids bodies and induced
oxidative membrane damage [23]. Furthermore, it blocked
protein synthesis of yeast cells [24] and inhibited repli-
cation of cytomegalovirus [25]. According to general use
of artesunate, the artemisinin resistance to malaria was
found in clinical trial [26]. The resistance gene mdr1,
cg10, tctp, and atp6 to artemisinin of Plasmodium cha-
baudi chabaudi were developed and transmitted to its
derivatives [27]. The pB2.3 resistant gene of N. fowleri
was appeared, thereby it located in mitochondria and
chromosome of the amoebae. In conclusion, one of the
drug treatment failure focused on ITS and pB2.3 genes of
N. fowleri.
This work was partially suppoted by: grant Number RGP 2552/02
from Department of Biology, Faculty of Science, Silpakorn University
at Sanamchan Palace, Nakhon Pathom, Thailand. We thank the head of
Department Parasitology, Faculty of Medicine Siriraj hospital, Mahidol
University for provided the research facility.
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