Sains Malaysiana 37(1): 107-113(2008)

Penindasan Terpilih Gen-Gen Staphylococcus aureus Rintang Metisilin

yang Dirawat dengan Ekstrak Metanol Melastoma malabathricum

(Selective Inhibition of Genes in Methicillin Resistant Staphylococcus Aureus (MRSA)

Treated with Melastoma Malabathricum Methanol Extract)

Zulaikah Mohamed

Makmal Kesihatan Awam

Jalan Kolam, Bukit Padang

Kota Kinabalu

88850 Sabah

Nazlina Ibrahim

Pusat Pengajian Biosains dan Bioteknologi

Fakulti Sains Dan Teknologi,

Universiti Kebangsaan Malaysia

Bangi, 43600, Selangor Darul Ehsan

Ismail Bin Ahmad

Jabatan Bioteknologi

Fakulti Sains dan Teknologi Sumber

UNIMAS, Kota Samarahan, Sarawak

Diserahkan:  Februari 2007/Diterima :  26 Jun 2007

ABSTRAK

Ekstrak metanol daun Melastoma malabathricum merencat pertumbuhan Staphylococcus aureus ATCC 25923 dan enam pencilan klinik Staphylococcus aureus rintang metisilin (MRSA 1-6). Nilai kepekatan terendah perencatan bakteria (MIC) adalah 1.565mg/ml dan nilai terendah bakterisid (MBC) adalah 3.125mg/ml. Ekstrak metanol daun M. malabathricum didapati merencat sistesis RNA MRSA4 pada kepekatan ekstrak 10mg/ml dengan mempamerkan profil RNA yang kehilangan tiga jalur berbanding kawalan. Analisis tindak balas transkriptase balik-berantai polimerase (RT-PCR) dengan tujuh pasang pencetus hanya berjaya menghasilkan empat amplikon cDNA. Kegagalan menghasilkan amplikon cDNA terhadap tiga pasang pencetus bagi gen-gen gyrA, femA dan nuc merujuk kepada kemungkinan berlakunya penindasan mRNA. Pemisahan protein endogen dan eksogen bakteria secara elektroforesis menunjukkan terdapatnya masing-masing tiga dan lima jalur protein yang ditindas dalam MRSA yang dirawat berbanding kawalan yang tidak dirawat. Manakala terdapat satu protein endogen dan tiga protein eksogen yang telah diekspres secara berlebihan.  Hasil analisis molekul dan proteom adalah selaras, dan berdasarkan pencetus yang digunakan, ekstrak metanol daun M. malabathricum berkemungkinan merencat pertumbuhan MRSA melalui penindasan sintesis DNA, penghasilan peptidoglikan dan penghasilan nuklease.

Kata kunci: Staphylococcus aureus Rintang Metisilin; Melastoma malabathricum; pengekspresan gen;  penghasilan protein

ABSTRACT

Methanol extract of Melastoma malabathricum leaves inhibited the growth of Staphylococcus aureus and six clinical isolates of Methicilin Resistant Stapyhlococcus aureus (MRSA 1-6). The minimum inhibitory concentration (MIC) of test substance was 1.565mg/ml and the minimum bacteriocidal concentration (MBC) was 3.125 mg/ml. The methanol extract suppressed RNA synthesis at 10 mg/ml as shown by RNA profile which was devoid of three bands compared to the control. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis using seven primer pairs was only successful in amplifying four cDNA amplicons. The failure to amplify three cDNA amplicons for three primer pairs corresponding to gyrA, femA and nuc genes, implied the possibility of suppression of the corresponding mRNA. Electrophoretic separation of endogenous and exogenuos bacterial proteins showed that three and five protein, respectively were not expressed. One endogenous and three exogenous proteins were over-expressed in treated MRSA compared with untreated control. The results of the molecular and proteomic analyses are in agreement, and based on primers being used, methanol extract of M. malabathricum leaves possibly inhibits MRSA growth through inhibition of DNA synthesis, peptidoglycan production, and nuclease production.

Keywords:  Methicillin resistant Staphylococcus aureus; Melastoma malabathricum; gene expression; protein production

RUJUKAN/REFERENCES

Barski, P., Piechowicz, L., Galin, J.S. & Kur, J.Z. 1996. Rapid assay for detection of methicillin-resistant Staphylococcus aureus using multiplex PCR.  Molecular and Cellular Probes 10: 471-475.

Black, C., Allan, I., Ford, S.K., Wilson, M. & Nab, R.M. 2004.  Biofilm-specific surface properties and protein expression in oral Streptococcus sanguis. Archives of Oral Biology 49: 295-304.

Burkill, I.H. 1966. A Dictionary of economic products of the Malay Peninsular. Ed. Ke 2.  The Ministry of Agriculture and Cooperative.  Kuala Lumpur, Malaysia: 1463-1465.

Corner, E.J.H. 1988.  Wayside tress of Malaya. Malayan Nature Society, Kuala Lumpur.

Cremonesi, P., Luzzana, M., Brasca, M., Stefano Morandi, S., Lodi, R., Vimercati, C., Agnellinia, D., Caramentid, G., Moroni, P. & Castiglioni, B. 2005.  Development of a multiplex PCR assay for the identification of Staphylococcus aureus enterotoxigenic strains isolated from milk and dairy products.  Molecular and Cellular Probes xx:1-7.

Eleaume, H. & Jabbouri, S. 2004.  Comparison of two standardisation methods in real-time quantitative RT-PCR to follow Staphylococcus aureus genes expression during in vitro growth.  Journal of Microbiological Methods 59:363– 370.

Frederick, M.A., Roger, B., Robert, E.K., David, D.M., Seidmer, J.G., Smith, J.A., & Struhl, K. 1987. Preparation and analysis of RNA dlm Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience: 4.1-4.10.

Gale, E.F, Reynolds, P.E. Richmond M.J & Waring, M.J. 1981.  The molecular basis of antibiotic action. London: John Wiley and Sons.

Gellert, M., Fisher, L.M & O'Dea, M.H. 1979. DNA gyrase: purification and catalytic properties of a fragment of gyrase B protein. Proceeding of National Academy Science, USA 76: 6289-6293.

Gellert, M., Mizuuchi, K., O'Dea, M.H. & Nash H.A. 1976.  DNA gyrase: an enzyme that introduced superhelical turns into DNA. Proceeding of National Academy Science, USA 73: 3872-3876.

Juana, V., Martín, L.,Oscar, D.G., Morales, M., Batista, N., Villar, J., Claverie, F.M., Sebastián, M.Á. 2004.  Simultaneous PCR detection of ica cluster and methicillin and mupirocin resistance genes in catheter-isolated Staphylococcus.  International Microbiology 7: 63–66.

Labischinski, H., Ehlert, K. & Berger, B.B. 1998. The targeting of factors necessary for expression of methicillin resistance in staphylococci. Journal of Antimicrobial Chemotherapy 41: 581–584.

Labischinski, H. & Johannsen, L. 1999. Cell wall targets in methicillin-resistant staphylococci. Drug Resistance Updates 2: 319–325.

Lachica, R.V.F., Hoeprich, P D. &. Riemann, H. P. 1972. Tolerance of staphylococcal thermonuclease to stress. Applied Microbiology 23: 994-997.

Lewis, K. 2000. Programmed death in bacteria. Microbiol. Mol. Biol. Rev. 64: 503-514.

Ling, B.D. & Berger, B.B. 1998. Increased overall antibiotic susceptibility in Staphylococcus aureus femAB null mutants. Antimicrobial Agents And Chemotherapy. 42: 936-938.

Lowy, F.D. 1998. Staphylococcus aureus infections. N. Engl. J. Med. 339: 520-532.

Madison, B. M. & Baselski, V. S. 1983. Rapid identification of Staphylococcus aureus in blood cultures by thermonuclease testing. Journal of  Clinical Microbiology 18: 722-724.

Matsuhashi, M., Song, M.D., Ishino, F., Wachi, M., Doi, M., Inoue, M., Ubukata, K., Yamashita, N. & Konno, M. 1986.  Molecular cloning of the gene of a penicillin-binding protein supposed to cause high resistance to b-lactam antibiotics in Staphylococcus aureus.  Journal of Bacteriology 167: 975-980

Mehrotra, M., Wang, G. & Johnson, W. M. J. 2000.  Multiplex PCR for Detection of Genes for Staphylococcus aureus Enterotoxins, Exfoliative Toxins, Toxic Shock Syndrome Toxin 1, and Methicillin Resistance. Journal of Clinical of Microbiology 38(3): 1032-1035.

Ng, E.Y, Trucksis M. & Hooper, D.C. 1996. Quinolone resistance mutations in topoisomerase IV: relationship of the flqA locus and genetic evidence that topoisomerase IV is the primary target and DNA gyrase the secondary target of fluoroquinolones in Staphylococcus aureus. Antimicrobial Agents Chemotherapy 40:1881-8.

Nurestri, S.M, Umi, K.S, Rohani, A.K. & Lim, P.K. 1995. Polar compounds from Melastoma malabathricum. Sains Malaysiana 24(2): 9-16.

Reynolds, P.E. &. Fuller, C. 1986.  Methicillin-resistant strains of Staphylococcus aureus, presence of identical additional penicilin-binding protein in all strains examined.  FEMS Microbiology Letter 33: 251-254.

Sato, K., Inoue, Y., Fujii, T., Aoyama, H., Inoue, M &  Mitsuhashi, S. 1986. Purification of and properties of DNA gyrase from a fluoroquinolone resistant strain of Escherichia coli.  Antimicrobial Agents Chemotherapy 30: 777-779.

Shen, L. L. & Pernet, A. G. 1985.  Mechanism of inhibition of DNA gyrase by analogues of nalidixic acid: the target of the drugs is DNA.  Proceeding of National Academy Science, USA 82: 307-311.

Singh, V.K., Jayaswal, R.K., & Wilkinson, B.J. 2001. Cell wall-active antibiotic induced proteins of Staphylococcus aureus identified using a proteomic approach. FEMS Microbiology Letters 199: 79-84.

Smith, R., Laudicinia, R.J. & Rufo, R.D. 1985. Learning Guides for Microbiology Laboratory, New York: John Wiley & Sons.

Song, M.D., Wachi, M., Doi, M., Ishino, F. & Matsuhashi, M. 1987.  Evolution of an inducible penicillin-target protein in methicillin-resistant Staphylococcus aureus by gene fusion.  FEBS Letter 221:9-22.

Takahata, M. & Nishino, T. 1988. DNA gyrase of Staphylococcus aureus and inhibitory effect of quinolones on its activity.  Antimicrobial Agents Chemotherapy 32:1192-1195.

Walsh, C. 2000.  Molecular component that confer antibacterial drug resistance.  Nature 406: 775-781

WHO, 2003.  Monitoring of antimicrobial resistance: report of an intercountry workshop. Vellore, Tamil Nadu, India.

Wong, K.C. 2004.  Some natural products from Melastoma malabathricum L.  Bulletin: The School of Chemical Sciences, Universiti Sains Malaysia 3(1):22-23.

Wu, W., Welsh, M.J., Kaufman, P.B. & Zhang, H.H. 1997.  Analysis of gene expression at the protein level. Dlm. Methods in Gene Biotechnology, New York: CRC Press, Boca Raton

Zulaikah M., Nazlina, I. & Ahmad, I.B. 2005. Methanolic and aqueous extracts of Melastoma malabathricum are effective against methicillin-resistant Staphylococcus aureus (MRSA). Proceedings of the 8th Symposium of Applied Biology; Marriot Putrajaya Hotel, Putrajaya, 22-23 June 2005. 691-694.

Zulaikah, M. 2007. Aktiviti perencatan ekstrak metanol daun Melastoma malabathricum terhadap Staphylococcus aureus rintang metisilin (MRSA). Tesis Sarjana. Universiti Kebangsaan Malaysia.