ITS and pB2.5 gene expression of Naegleria fowleri in drug resistance

doi:10.4236/health.2011.38088

Paper Menu >>

Journal Menu >>

Vol.3, No.8, 529-533 (2011) Health

doi:10.4236/health.2011.38088

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.

ABSTRACT

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

Resistance

1. INTRODUCTION

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. MATERIALS AND METHODS

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

530

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

CTG GTG ATG GTG TCT CTC ACA C-3′ and 5′́̀- CTC

TGA CAA TTT CTC TCT CAG TGG-3′. The amplicons

of amoebae were prepared from primers of ITS (ITS1;

5′́̀-GAACCTGCGTAGGGATCATTT-3′ and ITS2; 5′-

TTTCTTTTCCTCCC CTTATTA-3′) and pB2.3 (p3f; 5′-

GTGAAAACCTTTTTTCCATTTACA-3′) and p3r; 5′-

AAATAAAAA TTACCATTTGAAA-3′) by one – step

Super-script PCR (Invitrogen, Gransland). MW of each

amplicon was detected by 1.5% Agarose Gel Electro-

phoresis at 100 V for 30 min.

3. RESULTS AND DISCUSSION

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-

tesunate.

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

531

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

532

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.

4. ACKNOWLEDGEMENTS

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.

REFERENCES

[1] Hebbar, S., Bairy, I., Bhaskaranand, N., Upadhyaya, S.,

Sarma, M.S., Shetty, A.K.(2005) Fatal case of Naegleria

fowleri meningo-encephalitis in an infant: Case report.

Annals of Tropical Paediatrics, 25, 223-226.

doi:10.1179/146532805X58166

[2] Soltow, S.M. and Brenner, G.M. (2007) Synergistic ac-

tivities of azithromycin and amphotericin B against Nae-

gleria fowleri in vitro and in a mouse model of primary

amebic meningoencephalitis. Antimicrobial Agents and

Chemotherapy, 51, 23-27.

doi:10.1128/AAC.00788-06

[3] Donadio, S., Maffioli, S., Monciardini, P., Sosio, M. and

Jabes, D. (2010) Antibiotic discovery in the twenty-first

century: current trends and future perspectives. The

Journal of Antibiotics, 63, 423-430.

doi:10.1038/ja.2010.62

[4] Barker, K.S. and Rogers, P.D. (2006) Recent insights into

the mechanisms of antifungal resistance. Current Infe-

ctious Disease Reports, 8, 449-456.

doi:10.1007/s11908-006-0019-3

[5] Akins, R.A. (2005) An update on antifungal targets and

mechanisms of resistance in Candida albicans. Medical

Mycology, 43, 285-318.

doi:10.1080/13693780500138971

[6] Vermitsky, J.P. and Edlind, T.D. (2004) Azole resistance

in Candida glabrata: Coordinate upregulation of mul-

tidrug transporters and evidence for a Pdr1-like tran-

scription factor. Antimicrobial Agents and Chemotherapy,

48, 3773-3781. doi:10.1128/AAC.48.10.3773-3781.2004

[7] Tiewcharoen, S., Rabablert, J., Worawirunwong, D., Pra-

tumsrikajorn, T., Limsangurai, S. and Junnu, V. (2011)

Activity of chlorpromazine on nfa1 and Mp2CL5 genes

of Naegleria fowleri trophozoites. Health, 3, 166-177.

doi:10.4236/health.2011.33032

[8] Robinson, B.S, Monis, P.T. and Dobson, P.J (2006) Rapid,

sensitive, and discriminating identification of Naegle ri a

spp. by real-time PCR and melting-curve. Applied and

Environmental Microbiology, 72, 5857-5863.

doi:10.1128/AEM.00113-06

[9] Tsvetkova, N., Schild, M., Panaiotov, S., et al. (2004)

The identification of free-living environmental isolates of

amoebae from Bulgaria. Parasitology Research, 92, 405-

413.

[10] Tiewcharoen, S., Rabablert, J. and Junnu, V. (2009) In

vitro susceptibility of Naegleria fowleri trophozoites to,

amphotericin B-combined chlorpromazine. Research Jo-

urnal of Microbiology, 4, 320-333.

doi:10.3923/jm.2009.320.333

[11] Zhang, L., Zhang, Y., Zhoul, Y., et al. (2002) Response of

gene expression in Saccharomyces cerevisiae to am-

photericin B and nystatin measured by microarrays.

Journal Antimicrobial Chemotheapy, 49, 905-915.

doi:10.1093/jac/dkf001

[12] Manfredi, R., Fulgaro, C., Sabbatani, S., Legnani, G. and

Fasulo G. (2006) Emergence of amphotericin B-resi-

stant Cryptococcus laurentii meningoencephalitis shortly

after treatment for Cryptococcus neoformans meningitis

in a patient with aids. Aids Patient Care and STDs, 20,

227-232. doi:10.1089/apc.2006.20.227

[13] Orozco, E., Marchat, L.A, Gómez, C., López-Camarillo,

C. and Pérez, D.G. (2009) Drug resistance mechanisms in

Entamoeba histolytica, Giardia lamblia, Trichomonas

vaginalis, and opportunistic anaerobic protozoa. Antimi-

crobial Drug Resistance Infectious Disease, F, 549-559.

[14] Singh, A.K, Papadopoulou, B., Ouellette, M (2009) Gene

Amplification in Amphotericin B-Resistant Leishmania

tarentolae. Experimental Parasitology, 99, 141-147.

doi:10.1006/expr.2001.4663

[15] O'Shaughnessy, E.M, Lyman, C.A. and Walsh, T.J. (2009)

Amphotericin B: Polyene resistance mechanisms. Anti-

microbial Drug Resistance Infectious Disease, D, 295-

305.

[16] Mandell, G.L. and Coleman, E.J. (2000) Activities of

antimicrobial agents against intracellular pneumococci.

Antimicrobial Agents and Chemotherapy, 44, 2561-2563.

doi:10.1128/AAC.44.9.2561-2563.2000

[17] Mulet, X., Maciá, M.D., Mena, A., Juan, C., Pérez, J.L.,

Oliver, A. (2009) Azithromycin in Pseudomonas aerugi-

nosa biofilms: Bactericidal activity and selection of nfxB

mutants. Antimicrobial Agents and Chemotherapy, 53,

1552-1560. doi:10.1128/AAC.01264-08

[18] Galarza, P.G., Abad, R., Canigia, L.F, et al. (2010) New

mutation in 23S rRNA gene associated with high level of

azithromycin resistance in Neisseria gonorrhoeae. An-

timicrobial Agents and Chemotherapy, 54, 1652-1653.

[19] Gillis, R.J., White, K.G., Choi, K.H., et al. (2005) Mo-

lecular basis of azithromycin-resistant Pseudomonas

aeruginosa biofilms. Antimicrobial Agents and Chemo-

J. Rabablert et al. / Health 3 (2011) 529-533

533

therapy, 49, 3858-3867.

doi:10.1128/AAC.49.9.3858-3867.2005

[20] Chow, J.W. (2000) Aminoglycoside resistance in ente-

rococci. Clinical Infectious Diseases, 31, 586-589.

doi:10.1086/313949

[21] Livermore, D.M. (2002) Multiple mechanisms of antim-

icrobial resistance in Pseudomonas aeruginosa: Our

worst nightmare? Clinical Infectious Diseases, 34, 634-

640. doi:10.1086/338782

[22] Heuer, H., Krögerrecklenfort, E., Wellington, E.M., et al.

(2002) Gentamicin resistance genes in environmental

bacteria: Prevalence and transfer. FEMS Microbioogy

Ecology, 42, 289-302.

[23] Hartwig, C.L, Rosenthal, A.S, D'Angelo, J., et al. (2009)

Accumulation of artemisinin trioxane derivatives within

neutral lipids of Plasmodium falciparum malaria para-

sites is endoperoxide-dependent. Biochemistry Pharma-

cology, 77, 322-336. doi:10.1016/j.bcp.2008.10.015

[24] Li, W., Mo, W., Shen, D., et al. (2005) Yeast model un-

covers dual roles of mitochondria in the action of ar-

temisinin. PLoS Genetics, 1, 329.

doi:10.1371/journal.pgen.0010036

[25] Kaptein, S.J., Efferth, T., Leis, M., et al. (2006) The an-

ti-malaria drug artesunate inhibits replication of cy-

tomegalovirus in vitro and in vivo. Antiviral Research, 69,

60- 69. doi:10.1016/j.antiviral.2005.10.003

[26] Meshnick, S.R. (2002) Artemisinin: Mechanisms of ac-

tion, resistance and toxicity. International Journal for

Parasitology, 32, 1655-1660.

doi:10.1016/S0020-7519(02)00194-7

[27] Afonso, A., Hunt, P. , Cheesman, S., et al. (2006) Malaria

parasites can develop stable resistance to artemisinin but

lack mutations in candidate genes atp6 (Encoding the

sarcoplasmic and endoplasmic reticulum Ca2+ ATPase),

tctp, mdr1, and cg10. Antimicrobial Agents and Chemo-

therapy, 50, 480-489.

doi:10.1128/AAC.50.2.480-489.2006