3.MI691-Charis Amarantini Available online at http://jurnal.permi.or.id/index.php/mionline DOI: 10.5454/mi.7.1.3ISSN 1978-3477, eISSN 2087-8587 Vol 7, No 1, March 2013, p 17-23 *Corresponding author; Phone: +62-274-563929, Fax: +62- 274-513235, E-mail: charis@ukdw.ac.id Molecular techniques have become increasingly popular and potentially useful tool for the classification and identification of bacterial strains in most bacterial genera. One of which is 16S rDNA gene sequence analysis. The 16S rDNA gene is highly conserved within a species and among species of the same genus, and therefore it can be used as a reference for the speciation of bacteria (Woo et al. 2000). This method plays an important role in the identification of bacterial pathogens, which is useful not only for diagnosis, but also for phylogenetic classification. Most Salmonella strains are identified serologically as having O (somatic) and H (flagellar) antigens. There are, however, often serological cross-reaction between strains that phylogenetic classification is important to distinguish them from one another (Woo et al. 2001). Currently there are three known species in the Salmonella genus , Salmonella enterica, Salmonella bongori, and Salmonella subterranea (Shelobolina et al. 2004). Salmonella enterica comprises of six subspecies. They are S. enterica subsp. enterica (subsp. I), S. enterica subsp. salamae (subsp. II), S. enterica subsp. arizonae (IIIa subsp.), S. enterica subsp. diarizonae (subsp. IIIa), S. enterica subsp. houtenae (subsp. IV), and S. enterica subsp. indica (subsp. VI). Salmonella bongori was originally classified as S. enterica subspecies V. The majority of S. enterica subsp. enterica (subsp. I) cause most infections in humans and warm-blooded animals (Truper 2005; Tajbakhsh et al. 2011). Salmonella Typhi, the agent of typhoid fever, is included in subspecies I: S. enterica subsp. enterica serotype Typhi (S. enterica serovar Typhi; Salmonella A total of thirteen isolates representative of Salmonella Typhi from different geographical locations in Sumba Island, East Nusa Tenggara, Indonesia were identified by 16S rDNA gene sequences. Bacterial DNA TM extraction was prepared by using a Purelink Genomic DNA kit. The bacterial DNA and control were amplified using the specific primers for S. Typhi. These 16S rDNA gene sequence data were aligned with the corresponding available S. Typhi sequence and the reference organisms from the family Enterobacteriaceae from NCBI database by using the CLUSTAL X software. Phylogenetic trees were generated using the PHYLIP software package and the matrix of nucleotide similarity and nucleotide difference were generated by using the PHYDIT software. The results from the 16S rDNA analysis showed that the degree of similarity within these strains ranged from 99.13-100%. The percentage of sequence similarity between S. Typhi strains was very high (>99 %). Molecular phylogenetic analysis showed that all of the isolates formed a new center of diversity with S. Typhi T ATCC 19430 as a reference strain. Based on these results, all of the tested strains belonged to species of S. Typhi T suggested by their relatedness with the type strain of S. Typhi ATCC 19430 . Key words: 16S rDNA, phylogenetic analysis, Salmonella Typhi, typing Sebanyak 13 isolat Salmonella Typhi yang mewakili berbagai wilayah geografis di pulau Sumba, Nusa Tenggara Timur, Indonesia diidentifikasi berdasarkan sekuen gen 16S rDNA. DNA bakteri diekstraksi sesuai TM dengan petunjuk dari Purelink Genomic DNA kit. DNA bakteri dan kontrol diamplifikasi menggunakan primer spesifik untuk S. Typhi. Urutan basa sekuen gen 16S rDNA dianalisis bersama dengan sekuen 16S rDNA S. Typhi dan anggota famili Enterobacteriaceae yang diperoleh dari database NCBI menggunakan program ClustalX. Pohon filogeni dikonstruksi dengan menggunakan program PHYLIP sedangkan matriks similaritas dan perbedaan nukleotida dianalisis dengan menggunakan program PHYDIT. Hasil analisis sekuen 16S rDNA menunjukkan bahwa kemiripan di antara isolat S. Typhi berkisar antara 99,13-100%. Persentase kemiripan sekuen di antara isolat S. Typhi sangat tinggi (>99%). Analisis filogeni menunjukkan bahwa semua isolat T membentuk pusat keanekaragaman baru dengan isolat standard S. Typhi ATCC 19430 . Berdasarkan hasil tersebut, dapat disimpulkan bahwa semua isolat yang diuji tergolong dalam spesies S. Typhi yang ditunjukkan T dari kedekatan hubungan kekerabatan dengan isolat standard S. Typhi ATCC 19430 . Kata kunci: 16S rDNA, analisis filogenetik, Salmonella Typhi, typing 16S rDNA Typing of Salmonella Typhi Strains from Different Geographical Locations in Sumba Island East, Nusa Tenggara, Indonesia CHARIS AMARANTINI* AND TRI YAHYA BUDIARSO Biology Department, Universitas Kristen Duta Wacana, Jalan dr Wahidin Sudirohusodo 5-19, Yogyakarta 55224, Indonesia Typhi). Typhoid fever is most prevalent in tropical areas, including Indonesia (Moehario 2009). In Sumba Island, East Nusa Tenggara especially in Soutwest Sumba MATERIALS AND METHODS Extraction of Bacterial DNA, PCR Amplificati- on, and DNA Sequencing. Bacterial DNA was extracted in accordance with the protocol's instructions TM using a Purelink Genomic DNA Mini Kit (Invitrogen K1820-00). The bacterial DNA and control were st amplified with 25 pmol primers (1 Base Custom Oligos TM TM FBCO) and PCR SuperMix (illustra PuRe Taq TM Ready-To-Go PCR beads. PCR amplification for the 16S rDNA sequences showed bands of 428 bp, 484 bp, and 483 bp. These fragments were amplified using District over 197 cases or 725 infection/100.000 inhabitants were recorded in the database of the Karitas Hospital in 2006 (Amarantini et al. 2009). This number was higher than the average infection cases in rural areas in Indonesia, and it was nearly the same as the average cases in urban areas (810/100.000) according to World Health Organization (WHO) in 2003. These numbers suggested that the area was a good niche for fast growing microorganisms. Analysis of various S. Typhi strains using numerical systematic method showed that there were a big diversities in the use of the carbon sources by the S. Typhi isolates in these region (Amarantini et al. 2009). In addition to obtaining accurate data and strong discriminative ability to distinguish the strains, this study was aimed to identify and unravel the diversities of S. Typhi isolates from typhoid fever patients using molecular phylogenetic approach based on 16S rDNA gene sequences. Bacterial Strains. Thirteen isolates used in this research were isolated from the blood cultures of typhoid patients in Sumba Island. They came from different geographical regions in Karitas Hospital in Weetabula, a private clinic in Elopada Subdistrict in Southwest Sumba District, and Lendemoripa Hospital in Waikabubak in West Sumba District. Specimen collection methods were described in the journal article published previously (Amarantini et al. 2009). These isolates were identified using microbiological standard methods. All cultures were screened in Chromocult Coliform Agar (CCA). Typical colonies appear white and transparents due to the lack of ß- galactosidase and ß-glucoronidase enzymes. These colonies were confirmed using Triple Sugar Iron Agar (TSIA) (WHO 2003). All cultures were grown at 30 °C for 24 h on Brain Heart Infusion (BHI) agar before used. 18 AMARANTINI AND BUDIARSO Microbiol Indones primer R1 FR, R3 FR, and R5 FR respectively. The PCR product was gel purified with a QIAquick PCR purification kit (QIAgen, Hilden, Germany). The purified PCR product was sequenced with ABI Prism 3100-Avant Genetic Analyzer in accordance with the manufacture's instructions (Applied Biosystems, USA) using PCR primers. Analysis and Alignment of 16S rDNA Nucleotide Sequences. The 16S rDNA nucleotide sequences were analyzed, edited and assembled with Finch TV 1.4.0 and DNA Baser sequence analysis software. Complete assembled sequences were aligned with the corresponding S. Typhi sequences retrieved from the NCBI database with CUSTAL X software (Thompson et al. 1997). RESULTS The primers used to amplify these fragments are shown in Table 1 (Massi et al. 2005). The PCR mixtures were amplified for 40 cycles at 94 °C for 1 minute, 55 °C for 1 min, and 72 °C for 2 min, with a final extension at 72 °C for 10 min in automated Applied Biosystems GeneAmp PCR System 2400. An aliquot of 5 µL of each amplified product was electrophoresed in 3.0% (w/v) agarose gel, TM with a 100 bp DNA Ladder (Gene Ruler ). The PCR product was gel purified with a QIAquick PCR purification kit (QIAgen, Hilden, Germany). The purified PCR product was sequenced with ABI Prism 3100-Avant Genetic Analyzer according to the manufacturer's instructions (Applied Biosystems, USA) using the same primers as used in PCR. Construction of Phylogenetic Tree. Based on 16S rDNA nucleotide sequences, a phylogenetic tree was constructed with PHYLIP software package (Felsenstein 1993) with neighbor-joining algorithm (Saito and Nei 1987). The evolutionary distance matrix of the neighbor-joining method was generated according to the description from Jukes and Cantor (1969). The phylogenetic distances were obtained by adding only the values of the horizontal components. Eventually, the matrix of the nucleotide similarity and difference was generated with PHYDIT software (Chun 1999). Thirteen isolates used in this research came from different locations; seven isolates from East Wewewa, three isolates from Kodi, two isolates from North Wewewa, and one isolate from Waikabubak. These isolates were selected to represent their geographical origins. They're mapped according to the infected patient's place of residence based on global positioning system (Fig 1). Fig 1 Distribution of typhoid fever patients and 13 strains representation from different geograophic location in Sumba Island, East Nusa Tenggara. Tnumber Z47544 (S. Typhi ATCC 19430 ), AF029227 (S. bongori BR 1859), X80724.1 (Escherichia coli ATCC 25922), M59291 (Citrobacter freundii ATCC29935), M59160 (Serratia marcescens), M59149 (Erwinia carotovora ATCC 15713), X75275 (Yersinia ruckeri ATCC 29473), X75279 (Yersinia intermedia ER-3854), M59155 (Hafnia alvei ATCC 13337), X82248 (Photobacter luminescens DSM 3368), X82251 (Xenorhabdus nematophilus DSM 3370), X07652 (Proteus vulgaris IFAM 1731), and M59159 (Plesiomonas shigelloides) The phylogenetic analysis showed 13 representative strains of S. Typhi originated from different locations in Sumba Island, East Nusa Tenggara, which were then divided into three clades. The first clade consisted of seven strains. One isolate (HB01) originated from Wailabubu, North Kodi Subdistrict and six isolates were obtained from East Wewewa Subdistrict, which were HB05 (isolated from Wanowitu), HB09 (isolated from HB07 (isolated from Weerambo, East Wewewa), HB06 (isolated from Ombawawi North Wewewa), HB03 (isolated from Watubero Wailabubur North Kodi), and HB04 (isolated from Pakamutu, The third clade consisted of two strains. They were HB08 (isolated from Palekki/Mahaloko in North T Wewewa Subdistrict) and S. Typhi ATCC 19430 , which was the reference strain. The 16S rDNA nucleotide similarity values (%) and the number of nucleotide differences among 13 S. Typhi isolates from infected patients in different geographical locations in Sumba Island and the T reference strain S. Typhi ATCC 19430 are shown in Table 1 and 2. The sequences of the 13 strains and S. T Typhi ATCC 19430 showed >99% similarity. It was also evident that strain HB10 was identical with the strain HB09, and the strain HB11 was identical with the strains HB09 and HB10 (Table 2). The two other strains (HB03 and HB04) were also identicals (Table 3). Bondokodi Kodi). Katikutana Kodi Kodi Bangedo North Kodi Waikabubak Lamboya Laura Loli Mamboro Tana Righu Umbu Ratu Nggay West Umbu Ratu Nggay Wanokaka West Wawewa South Wawewa East Wawewa 119'00' 119'10' 119'20' 119'30' 119'40' 119'50' 9 '2 0 ' 9 '3 0 ' 9 '4 0 ' 9 '5 0 ' 119'00' 119'10' 119'20' 119'30' 119'40' 119'50' 9 '2 0 ' 9 '3 0 ' 9 '4 0 ' 9 '5 0 ' Strain representation Patient residence Phylogenetic analysis results of 13 S. Typhi isolates based on 16S rDNA gene sequences are shown in Fig 2. The 16s rDNA gene sequences of these isolates were initially compared to those of the Enterobacteriaceae family reported in the Gene Bank with the accesion Omba Rade), HB 10 (isolated from Weedindi), HB11 (isolated from Durru Lodo), HB12 (isolated from Kongge), and HB13 (isolated from Elopada). The second clade consisted of five strains, which were HB02 (isolated from Kampung Sawah Waikabubak), Volume 7, 2013 Microbiol Indones 19 Table 2 16S rDNA similarity values (%) and the number of nucleotide differences between seven strains and the reference T strains of S.Typhi ATCC 19430 within the first clade 20 AMARANTINI AND BUDIARSO Microbiol Indones Primer Sequence Nucleotide position R1F 5' AGTTTGATCCTGGCTCAG 3'* 3-20 (AC: Z47544) R1R 5' AGTACTTTACAACCCGAAGG 3'* 411-430 (AC: Z47544) R3F 5' AAGTACTTTCAGCGGGGA 3'* 424-441 (AC: Z47544) R3R 5' TTGAGTTTTAACCTTGCGG 3'* 898-916 (AC: Z47544) R5F 5' AACTCAAATGAATTGACGG 3'* 901-919 (AC: Z47544) R5R 5' AGGCCCGGGAACGTATTCAC 3'* 1364-1383 (AC: Z47544) Table 1 The primers used for PCR amplification of 16S rDNA gene sequence of Salmonella Typhi HB09 HB10 HB11 HB12 HB01 HB13 HB05 S. Typhi ATCC T 19430 S. bongori BR1859 HB09 --- 0/1383 0/1381 7/1382 4/1383 1/1382 1/1381 5/1381 33/1377 HB10 100.00 --- 0/1381 7/1381 3/1382 1/1382 1/1381 5/1381 33/1377 HB11 100.00 100.00 --- 7/1381 3/1381 1/1381 1/1381 5/1381 33/1377 HB12 99.49 99.49 99.49 --- 11/1382 8/1381 8/1381 12/1381 40/1377 HB01 99.71 99.78 99.78 99.20 --- 4/1382 4/1381 8/1381 36/1377 HB13 99.93 99.93 99.93 99.42 99.71 2/1381 6/1381 34/1377 HB05 99.93 99.93 99.93 99.42 99.71 99.86 --- 4/1381 32/1377 99.64 99.64 99.64 99.13 99.42 99.57 99.71 --- 36/1497 S. bongori BR1859 97.60 97.60 97.60 97.10 97.39 97.53 97.68 97.60 --- S. Typhi ATCC T 19430 HB03 HB04 HB06 HB02 HB07 HB08 S. Typhi ATCC T 19430 S. bongori BR1859 HB03 --- 0/1384 5/1381 5/1381 6/1381 4/1381 7/1381 35/1377 HB04 100.00 --- 5/1381 5/1381 6/1381 4/1381 7/1381 35/1377 HB06 99.64 99.64 --- 4/1381 5/1381 3/1381 6/1381 34/1377 HB02 99.64 99.64 99.71 --- 1/1382 3/1381 6/1381 34/1377 HB07 99.57 99.57 99.64 99.93 --- 4/1381 7/1381 35/1377 HB08 99.71 99.71 99.78 99.78 99.71 --- 3/1381 33/1377 99.49 99.49 99.57 99.57 99.49 99.78 --- 36/1497 97.46 97.46 97.53 97.53 97.46 97.60 97.60 --- S. Typhi ATCC T 19430 S. bongori BR1859 Table 3 16S rRNA similarity values (%) and the number of nucleotide differences between six strains and the reference strains T of S.Typhi ATCC 19430 within the two and the third clades AC: GenBank accession no. *Reference: Massi et al. 2005 0.01 T reference strain S. Typhi ATCC 19430 . It was also shown that strains within the species have diverse 16S rDNA gene sequences. In fact, all of the strains fall into three clades. These clades demostrated that genetic diversity of the tested strains could be unraveled using phylogenetic tree based on 16S rDNA sequences. It is shown in phylogenetic tree that each clade is made up of strains from different geographical areas in the Sumba Island especially in Southwest Sumba District. The six tested strains within the first clade Volume 7, 2013 Microbiol Indones 21 DISCUSSION entatives of the genus Salmonella clearly showed that all of the tested strains form a new center T of diversity with S. Typhi ATCC 19430 (Fig 2). This result directly proved that all of the isolates belong to S. Typhi species because of their relationships with the Phylogenetic analysis based on comparison of 16S rDNA nucleotide sequences of the 13 strains with the corresponding nucleotide sequence of available repres Fig 2 Neighbour-joining phylogeny tree constructed on the basis of 16S rDNA gene sequences showing relationship amongst the thirteen representatives of S. Typhi isolates from the different locations in Sumba Island, East Nusa Tenggara. The arrow indicates estimated root position of the three as determined using Plesiomonas shigelloides (M59159) as an outgroup. Bar, 1 substitution per 100 nucleotides. indicated that these strains are indigenous from the Southwest Sumba District. ACKNOWLEDGMENTS This research was supported by The Directorate General of Higher Education, Department of National Education (Hibah Bersaing) 2012, contract no: 560.7/K5/KL/2012 date 10-02-2012. A special gratitude is given to Karitas Hospital in Weetabula in Southwest Sumba District and Lende Moripa Hospital in Waikabubak in West Sumba District East Nusa Tenggara for their assistance in collecting the samples. I should also thank Sr. Sili Bouka ADM-the Director of Karitas Hospital, dr. Loeta Lapoe Moekoe-the Director of Lende Moripa Hospital and all doctors of Karitas Hospital for their assistance during the research. REFERENCES Amarantini C, Sembiring L, Kushadiwijaya H, Asmara W. 2009. Seleksi bakteri Salmonella Typhi dari kultur darah penderita demam tifoid [Selection of Samonella Typhi bacteria from the blood culture of typhoid patients]. In: Wijaya A, Darmawan D, Tuti R, Atmanto T, Nurohman S, editors. Revitalisasi MIPA dan Pendidikan MIPA dalam Rangka Penguatan Kapasitas Kelembagaan dan Profesionalisme Menuju World Class University. Proceeding of National Seminar on Research, Education, and Applied of Mathematics and Natural Sciences; 2009 May 16. Yogyakarta(ID): p B13-B20. Amarantini C, Sembiring L, Kushadiwijaya H, Asmara W. 2009. Isolasi, karakterisasi dan pengelompokan strain Salmonella Typhi asal kabupaten sumba barat daya nusa tenggara timur berdasarkan sifat-sifat fenotip [Isolation, characterization and grouping of Salmonella Typhi strains in the Southwest Sumba Regency East Nusa Tenggara based on phenotypic characteristics. Berkala Penelitian Hayati 14(2):191-196. Baumler AJ, Tsolis RM, Ficht TA, Adams LG. 1998. Evolution of host adaptation in Salmonella enterica. Infect Immun. 66(10):4579-4587. Christensen H, Nordentoft S, Olsen JE. 1998. Phylogenetic relationship of Salmonella based on rRNA sequences. Int J Syst Bacteriol. 48(2):605-610. doi:10.1099/00207713- 48-2-605. Chun J. 1999. Phylogenetic Editor (PHYDIT). Windows Version. Drancourt M, Bollet C, Carlioz A, Martelin R, Gayral JP, Raoult D. 2000. 16S Ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. J Clin Microbiol. 38(10): 3623-3630. Felsenstein J. 1993. Phylogeny inference package version 3,5c. Departement of Genetics. Seattle (USA): University of Washington. were derived from East Wewewa Subdistrict, whereas the other isolates were derived from North Kodi Subdistrict. The second clade consisted of two strains originally from Kodi, one strain derived from Waikabubak, and the two strains derived from East Wewewa and North Wewewa. Based on the coordinate position of the infected patient's residences (Fig 1), we learned how the typhoid fever spreads among the inhabitants. Most of the patients lived in East Wewewa Subdistrict. It appears that the strains were distributed in finger pattern to nearby subdistrict such as North Wewewa, South Wewewa, West Wewewa, and then goes further out to the Kodi Bangedo, Kodi, and North Kodi. Therefore, these results indirectly showed that there were inter geographical distribution of the strains due to migration of people in this area. Identification to the species level requires that the tested strains 16S rDNA sequence has 99% similarity with the sequence of the reference strain in GenBank (Drancourt et al. 2000). It is shown in this study that the 16S rDNA sequence of these 13 isolates had >99% similarity with the sequence of the closest strain in GenBank. Thus these isolates were identified as S. Typhi. In terms of nucleotide similarity and nucleotide differences (Table 1), it was shown that the strain HB10 was identical with HB09, and the strain HB11 was identical with HB09 and HB10. However, this result did not entirely coherent with its phylogenetic relatedness based on phylogenetic tree. It is apparent in Fig 2 that the closest strain was found to be the strain HB12. Two tested strains, HB03, and HB 04 (Table 2), were fully congruent in their similarity values, nucleotide differences and phylogenetic analysis. It is especially interesting that the strain HB 08 from Palekki/Mahaloko North Wewewa T was found to be closely related to S.Typhi ATCC 19430 . Based on analysis of housekeeping and rRNA genes, it has been understood that the genus Salmonella contains two lineages that had diverged considerably from each other during evolution. These lineages represent two distinct species, S. enterica and S. bongori (Baumler et al. 1998). Christensen et al. (1998) also demostrated that the analysis of 16S rDNA sequences separated S. bongori from S. enterica, and these two species from the complex of E. coli and Shigella species. This corresponds to the topology of this phylogeny tree (Fig 2). In summary, all the isolates were identified as S. Typhi species because of their association with the type T strain of S. Typhi ATCC 19430 . The fact that the 13 isolates belonging to S. Typhi species formed a new center of diversity within the 16S rDNA gene tree Microbiol Indones22 AMARANTINI AND BUDIARSO Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. 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Identification and sequencing of Salmonella enterica serotype Typhi isolates obtained from patients with perforation and non-perforation typhoid fever. Southeast Asian J Trop Med Publ Health. 36(1):118- 122. Moehario LM. 2009. The molecular epidemiology of Salmonella Typhi across Indonesia reveals bacterial migration. J Infect Dev Ctries. 3(8):579-584. doi:10.3855/jidc.548. Saitou N, Nei M. 1987. The Neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 4(4):406-425. Tajbakhsh M, Nayer BN, Motavaze K, Kharaziha P, Chiani M, Zali MR, Klena JD. 2011. Phylogenetic relationship of Salmonella enterica strains in Tehran Iran using 16S rRNA and gyrB gene sequences. J Infect Dev Ctries. 5(6): 465-472. doi:10.3855/jidc.1504. Volume 7, 2013 Microbiol Indones 23