Sains Malaysiana 47(8)(2018): 1795–1800

http://dx.doi.org/10.17576/jsm-2018-4708-18

 

Detection of Leptospira Species in Environmental Samples by Amplification of 16S rRNA and rpoβ Genes

(Pengesanan Spesies Leptospira dalam Sampel Alam Sekitar melalui 16S rRNA dan Gen rpoβ Diperkuat)

 

NURUL FARHANA ZULKIFLI¹, STEFFI JULAN WAN¹, VASANTHA KUMARI NEELA², HUI YEE CHEE², SITI NORBAYA MASRI², MAZEN M. JAMIL AL-OBAIDI¹ & MOHD NASIR MOHD DESA^1,3*

 

¹Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

 

²Department of Medical Microbiology and Parasitology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

 

³Laboratory of Halal Science Research, Halal Products Research Institute, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

 

Received: 8 Febuary 2018/Accepted: 23 April 2018

 

ABSTRACT

This study attempted to identify and determine distribution of Leptospira spp. in environmental samples using 16S rRNA and rpoβ genes amplification. The samples were collected from high risk areas in Selangor, Malaysia. A total of 105 environmental samples consisting of soil and water were subjected to direct DNA extraction and PCR reaction. PCR products were analysed using gel electrophoresis and subjected to sequence analysis. Thirteen out of 105 (12.38%) samples were amplified for 16S rRNA with an expected amplicon size of 330 bp, while 50 out of 105 (47.62%) samples showed amplification using rpoβ primers, but were not of expected size. Of the 13 16S rRNA amplified samples, only 5 were identified as Leptospira in the gene sequence analysis and clustered under uncultured group via phylogenetic tree. This study showed the DNA-based approach using PCR and sequence analysis is able to detect the presence of Leptospira, although environmental samples may contain diverse microbial populations that may complicate the detection. Overall, the study suggested the importance of surveillance for Leptospira from environmental samples.

 

Keywords: Environment; Leptospira; rpoβ; Selangor;16S rRNA

 

ABSTRAK

Kajian ini dijalankan untuk mengenal pasti dan menentukan penyebaran Leptospira spp. dalam sampel alam sekitar menggunakan 16S rNA dan gen rpoβ diperkuat. Sampel diperoleh daripada kawasan yang mempunyai risiko tinggi di Selangor, Malaysia. Sampel keseluruhan adalah 105 yang terdiri daripada tanah dan air yang tertakluk secara langsung kepada pengekstrakan DNA dan reaksi PCR. Produk PCR dianalisis menggunakan elektroforesis gel dan tertakluk kepada analisis urutan. Tiga belas sampel daripada 105 (12.38%) menunjukkan amplifikasi untuk 16S rRNA dengan saiz amplikon terjangka 330 bp, manakala 50 sampel daripada 105 (47.62%) menunjukkan sampel amplikasi menggunakan pencetus rpoβ, tetapi tidak mengikut saiz yang dijangka. Daripada 13 16S rRNA sampel diperkuat, hanya 5 dikenal pasti sebagai Leptospira dalam urutan gen analisis dan berkelompok di bawah kumpulan tidak dibiak melalui pohon filogenetik. Kajian ini menunjukkan pendekatan berasaskan DNA menggunakan PCR dan analisis urutan dapat mengesan kehadiran Leptospira, walaupun sampel alam sekitar berkemungkinan mengandungi pelbagai populasi mikrob yang boleh merumitkan pengesanan. Secara keseluruhan, kajian ini mencadangkan kepentingan pengawasan Leptospira daripada sampel alam sekitar.

 

Kata kunci: Alam sekitar; Leptospira; rpoβ; Selangor; 16S rRNA

REFERENCES

Adler, B. & Moctezuma, A.D.L.P. 2010. Leptospira and leptospirosis. Veterinary Microbiology 140(3-4): 287-296.

Case, R.J., Boucher, Y., Dahllöf, I., Holmstrom, C. & Kjelleberg, S. 2007. Use of 16S rRNA and rpoβ genes as molecular markers for microbial ecology studies. Applied Environmental Microbiology 73: 278-288.

Ganoza, C.A., Matthias, M.A., Richards, D.C., Brouwer, K.C., Cunningham, C.B., Segura, E.R., Gilman, R.H., Gotuzzo, E. & Vinetz, J.M. 2006. Determining risk for severe leptospirosis by molecular analysis of environmental surface waters for pathogenic leptospira. PLOS Medicine 3: 1329-1340.

Gabor, E.M., Vries, E.J.D. & Janssen, D.B. 2003. Efficient recovery of environmental DNA for expression cloning by indirect extraction methods. FEMS Microbiology Ecology 44(22): 153-163.

Garba, B., Bahaman, A.R., Khairani-Bejo, S., Zakaria, Z. & Mutalib, A.A. 2017. Retrospective study of leptospirosis in malaysia. Ecohealth 14: 389-398.

Haake, D.A. & Levett, P.N. 2015. Leptospirosis in humans. Current Topics in Microbiology Immunology 387: 65-97.

Ismail, S., Wahab, Z., Badya, N., Rahman, N.I.A., Haque, M., Yeo, C.C. & Latif, A.Z.A. 2014. A study on the presence of pathogenic Leptospira spp. in environmental water samples obtained from selected recreational areas in terengganu, malaysia. Research Journal of Pharmacy and Technology 7(10): 1153-1157.

Lim, J.K., Vikneswaran, A., Murugaiyah, A.S.R., Rahman, H.A., Mohamed, N.S., Shamsudin, N.N. & Tan, J.C. 2011. A case study: Leptospirosis in Malaysia. WebmedCentral Infectious Diseases 2(12): WMC002764.

Liu, J., Wu, W., Chen, C., Sun, F. & Chen, Y. 2011. Prokaryotic diversity, composition structure, and phylogenetic analysis of microbial communities in leachate sediment ecosystems. Applied Microbiology Biotechnology 91(6): 1659-1675.

Merien, F., Amouriaux, P., Perolat, P., Baranton, G. & Girons, I.S. 1992. Polymerase chain reaction for detection of leptospira in clinical samples. Journal of Clinical Microbiology 30(9): 2219-2224.

Mwachui, M.A., Crump, L., Hartskeerl, R., Zisstag, J. & Hattendorf, J. 2015. Environmental and behavioural determinants of leptospirosis transmission: A systematic review. PLOS Neglected Tropical Diseases 9(9): e0003843.

Scola, B.L., Bui, L.T.M., Baranton, G., Khamis, A. & Raoult, D. 2006. Partial rpoβ gene sequencing for identification of Leptospira species. FEMS Microbiology Letters 263(2): 142-147.

Thaipadungpanit, J., Wuthiekanun, V., Chantratita, N., Yimsamran, S., Amornchai, P., Boonslip, S., Maneeboonyang, W., Tharnpoophasiam, P., Saiprom, N., Mahakunkijcharoen, Y., Day, N.P.J., Singhasivanon, P., Peacock, S.J. & Limmathurotsakul, D. 2013. Short report: Leptospira species in floodwater during the 2011 floods in the bangkok metropolitan region, Thailand. The American Journal of Tropical Medicine and Hygiene 89(4): 794-796.

 

*Corresponding author; email: mnasir@upm.edu.my