01. Charis (Molecular).cdr Molecular Phylogeny of Salmonellae: Relationships among Salmonella Species Determined from gyrA, gyrB, parC, and parE Genes CHARIS AMARANTINI* AND DHIRA SATWIKA Faculty of Biotechnology, Duta Wacana Christian University Jalan dr. Wahidin Sudirohusodo 5-19, Yogyakarta 55224, Indonesia Study on molecular characteristics of Salmonella from clinical isolates was done in order to find out its relationship, especially those isolated from Indonesia. Partial sequence of genes belonging to QRDR region, i.e. gyrA, gyrB, parC, and parE were employed. Specific primer pairs covering those genes are used to amplify the bacterial DNA obtained. The amplicons were then analyzed by means of sequencing, and the sequences are analysed bioinformatically to find out similarities and build phylogenetic trees. By comparing all of the phylogenetic tree from QRDR region, this study revealed gyrA as the most suitable gene for rapidly identify member of salmonellae as it gives better separation of samples being analysed. However, the use of parC is recommended as it gives a consistent and reliable value to separate member of Salmonella and other Enterobacter. Further studies are under way to include member of this group, like E. coli, and the use of full sequence of QRDR genes region to verify this report. Key words: gyrA, parC, QRDR, Salmonella Penelitian ini dilakukan untuk mencari kekerabatan dan hubungan filogeni Salmonella yang berasal dari isolat klinis yang diisolasi dari Indonesia. Selama ini sudah dilaporkan pemanfaatan gen-gen yang berada pada daerah QRDR dapat digunakan sebagai penanda untuk menentukan hubungan kekerabatan tersebut. Partial sequence gen-gen pada daerah tersebut, yaitu gyrA, gyrB, parC dan parE digunakan untuk tujuan tersebut. Pasangan primer yang mengamplifikasi urutan parsial setiap gen digunakan dalam reaksi PCR, selanjutnya hasil amplifikasi DNA isolat sampel dianalisa dengan melakukan sekuensing. Setelah urutan parsial DNA setiap gen diketahui, dilakukan analisa secara bioinformatis dengan melakukan alignment DNA sampel dengan database yang tersedia. Analisa ini memberi data berupa indeks kesamaan nukleotida hingga pembuatan pohon filogeni, yang dapat digunakan untuk melacak dan mencari kekerabatan setiap isolat. Hasil penelitian menunjukkan bahwa gyrA merupakan gen yang dapat digunakan untuk identifikasi cepat anggota Salmonella karena memberi resolusi paling baik. Meskipun demikian, parC merupakan gen yang direkomendasikan untuk memisahkan anggota Salmonella maupun kelompok bakteri yang erat kekerabatannya, misal kelompok Enterobacter, karena konsistensi pemisahan yang baik. Kata kunci : gyrA, parC, QRDR, Salmonella *Corresponding author; Phone: +62-274-563929, Fax: +62- 274-513235, E-mail: charis@staff.ukdw.ac.id Salmonellae are a diverse group of gram-negative bacteria and consist of a number closely related organisms belonging to the family Enterobacteriaceae. For a long time, the taxonomic classification and nomenclature of this group changed several times. The last classification systems have been validly proposed for two-species systems, Salmonella enterica and Salmonella bongori (Chang et al. 1997). Currently, Shelobolina et al. (2004) discovered Salmonella subterranea sp. nov. as a new species belongs to the genus Salmonella. In addition to the taxonomic classification of two- species systems, the salmonellae were categorized by serotype. There are over 2,500 serotypes associated with gastroenteritis and typhoid fever in human. The majority of these serotypes are often extremely difficult to be separated based on its biochemical characteristics. Therefore, it would be useful to classify these groups based on molecular typing methods. Molecular phylogenetic approach for classi- fication of salmonellae is important due to the increased spread of Salmonella strains, especially Salmonella enterica serovar Typhi (S. Typhi) with fluor oquinol one reduced susceptibility. Reports already available mentioning the use of genetic traits to differentiate this bacteria (for example: Tajbakhsh et al. 2011, Muthu et al. 2014). Nalidixicacid resistant of S. Typhi (NARST) strains with decreased susceptibility -1 to ciprofloxacin (0.125-1 µgL ) becomes a major problem in the Indian subcontinent (Capoor et al. 2007), and molecular analysis showed mutations of Vol.9, No.1, March 2015, p 1-8 DOI: 10.5454/mi.9.1.1 some genes in quinolone resistance-determining region (QRDR). It was reported, a point mutation in QRDR of the gyrA gene at various sites, especially at codons coding for serine at position 83 and aspartate at position 87 (Afzal et al. 2013). Other mutation was observed in the genes coding for DNA gyrase (gyrA or gyrB) or topoisomerase (parC and parE) in the resistant strain (Muthu et al. 2014). It was reported earlier some S. Typhi strains are resistant to nalidixic acid. The taxonomic classification of these strains based on 16S rRNA gene showed the sensitive and resistant isolates can not be separated into different clades (Amarantini and Budiarso 2013). D e t a i l e d a n a l y s i s f o r b a c t e r i a l p h y l o g e n e t i c relationships through the QRDR of the gyrA and gyrB subunits of DNA gyrase and the parC and parE subunits of topoisomerase IV might be possible for a better classification than 16S rRNA regions. The present study was done to assess the use of gyrA and gyrB genes in QRDR and parC and parE genes in topoisomerase IV for determining the phylogenetic relationship among sensitive and resistant S. Typhi isolates. MATERIALS AND METHODS Bacterial Strains. Four isolates used in this study were obtained from the previous research (Amarantini and Budiarso 2013); i.e. two nalidixic acid-resistant and two nalidixic acid-sensitive isolates. Two isolates from reference collection of PT Biofarma (S. Typhi NCTC 786) and BLK Yogyakarta (S. Typhi O) were also included. DNA Isolation. All isolates were cultured in Brain Heart Infusion Broth at 37 °C for 18 h prior to DNA extraction. To isolate chromosomal DNA, 1 ml of overnight culture were put into a 1.5 mL centrifugetube and were centrifuged at 5000 rpm, 15 min to obtain the cells. Isolation of DNA was carried out in accordance with the phenol-chloroform-isoamyl alcohol method (Sambrook et al. 1989). PCR Experiments of QRDRs of the gyrA, gyrB, parC, and parE Genes of S. Typhi. DNA isolated from bacterial strains were amplified by PCR using spesific primer (Table 1) for gyrA, gyrB, parC, and parE (Ling et al. 2003). The PCR was performed using TM DreamTaq Green PCR Master Mix in total reaction TM volume of 50µL containing 25µL of 2x DreamTaq Green PCR Master Mix, 1 µL of 1 µM primer stocks, and 1 µL of template DNA. The PCR reaction mixtures were amplified for 35 cycles with initial-denaturation at 95 ºC for 5 min, denaturation at 94 ºC for 1 min, annealing at 52 ºC for 1 min, and extention at 72 ºC for 1 min, with a final extension at 72 ºC for 5 min. An aliquot of 5 μL of each amplified product was electrophorese in 1.2% (wt/vol) agarose gel using 1x TBE buffer gel stained with Sybr® Safe DNA stain (Life technologies). A 100-bp DNA ladder (Fermentas, Germany) were included as molecular weight marker. DNA Sequencing and Phylogenetic Analysis. PCR products were purified and sequenced by outsourcing the samples to Macrogen Inc, Korea. The nucleotide sequences were edited and assembled using SeqMan and EditSeq (DNA Star, Laser Gene 6, M a d i s o n , W I , U S A ) . P h y l o g e n e t i c t r e e w a s constructed with Mega v5 (Tamura et al. 2011) with the neighbor-joining algorithm (Saito and Nei 1987). The evolutionary distance matrix for the neighbor- joining method was generated in accordance with the description introduced by Jukes and Cantor (1969). T h e m a t r i x o f t h e n u c l e o t i d e s i m i l a r i t y a n d difference was generated with PHYDIT software (Chun 1999). RESULTS We have amplified and sequenced two DNA fragments of S. Typhi containing gyrA and gyrB QRDRs. We also used two pairs of primers to amplify Table 1 Primers used to sequence gyrB/gyrA and parE/C (Ling et al. 2003) Primer name gyrA gyrB parC parE 2 AMARANTINI Microbiol Indones Primer sequence Size (bp) F, 5’ -TgTCCGAGATGGCCTGAAGC -3' 347 R, 5'-TACCGTCATAGTTATCCACG -3' F, 5' -CAAACTGGCGGACTGTCAGG-3' 345 R, 5'-TTCCGGCATCTGACGATAGA-3' F, 5' ATGAGCGATATGGCAGAGCG 3' 412 R, 5' TGACCGAGTTCGCTTAACAG 3' F, 5' GACCGAGCTGTTCCTTGTGG 3' 272 R, 5' AGCAGAGTAGCGATATGCAA 3' the parC and parE Q R D Rs. As expected, two amplification products of 435 and 297 bp were obtanined and their nucleotide sequences were determined. From these sequence data, we construct the phylogenetic structure and determine the genetic relationship among the nalidixic acid-sensitive and - resistant isolates. As shown in Figure 1, the phylogenetic analysis determined by gyrA sequence clearly separate the sensitive and resistant isolates into different clusters. Two resistant isolates (BPE 127.1 MC and BPE 122.4 CCA) were delineated using the gyrA gene. The percentage similarity within each strains ranged from 73.25-93.35% (Table 2). In contrast to the gyrA dendogram, the phylogenetic structure of Salmonella on the basis of gyrB gene sequences (345 bp) were not able to distinguish the sensitive and resistant isolates (Fig 2). The resistant isolates were grouped as the same cluster with the sensitive isolates. Unlike the gyrA dendogram, the similarity percentage between each Samonella Typhi O (BLK Yogyakarta) Samonella Typhi BPE 122.1 CCA Samonella Typhi BPE 122.4 CCA R* Samonella Typhi BPE 127.1 MC R* Samonella Typhi NCTC 786 Samonella Typhi RSK 5.1 SSA Salmonella Newport strain SN71 (KC121321.1) Salmonella Typhi strain ST73 (HQ176366.1) Escherichia coli(AY323807.1) SalmonellaTyphi strain ST80 (HQ176368.1) 13 100 55 22 41 52 0.1 Fig 1 Phylogenetic tree of Salmonella strains based on gyrA sequences analysis. The tree was constructed by neighbor- joining method. Bar, 1 substitution per 10 nucleotides. Table 2 Nucleotide similarity values (%) and the number of nucleotide differences between Salmonella strains based on gyrA sequence S . T y p h i O ( B L K Y o g y ak ar ta ) S .T y p h i B P E 1 2 2 .1 C C A S .T y p h i B P E 1 2 2 .4 C C A R * S . T y p h i B P E 1 2 7 .1 M C R * S . T y p h i N C T C 7 8 6 S . T y p h i R S K 5 .1 S S A H Q 1 7 6 3 6 8 .1 H Q 1 7 6 3 6 6 .1 K C 1 2 1 3 2 1 .1 A Y 3 2 3 8 0 7 .1 S. Typhi O (BLK Yogyakarta) --- 25/340 23/346 30/343 38/331 49/350 192/335 192/335 158/284 136/232 S.Typhi BPE 122.1 CCA 92.65 --- 41/363 38/355 33/325 126/471 249/423 249/423 155/284 132/232 S.Typhi BPE 122.4 CCA R* 93.35 88.71 --- 25/358 32/329 67/370 202/356 202/356 156/283 135/232 S. Typhi BPE 127.1 MC R* 91.25 89.30 93.02 --- 25/328 51/359 194/348 194/348 153/283 134/232 S. Typhi NCTC 786 88.52 89.85 90.27 92.38 --- 39/330 178/324 178/324 154/282 131/228 S. Typhi RSK 5.1 SSA 86.00 73.25 81.89 85.79 88.18 --- 262/427 262/427 154/284 132/232 HQ176368.1 42.69 41.13 43.26 44.25 45.06 38.64 --- 0/434 3/290 16/238 HQ176366.1 42.69 41.13 43.26 44.25 45.06 38.64 100.00 --- 3/290 16/238 KC121321.1 44.37 45.42 44.88 45.94 45.39 45.77 98.97 98.97 --- 18/235 AY323807.1 41.38 43.10 41.81 42.24 42.54 43.10 93.28 93.28 92.34 --- Volume 9, 2015 Microbiol Indones 3 Samonella Typhi BPE 127.1 MC R* Samonella Typhi BPE 122.4 CCA R* Samonella Typhi O (BLK Yogyakarta) Samonella Typhi RSK 5.1 SSA Samonella Typhi NCTC 786 Escherichia coli strain 408/65-1(DQ447149.1) Burkholderia cepaciaLMG1222 (AY996867.1) Salmonella Typhi strain 208 (EF064853.1) Salmonella Typhi strain ATCC 19430 (AY370864.1) 100 99 0.1 Fig 2 Phylogenetic tree of Salmonella strains based on gyrB sequences analysis. The tree was constructed by neighbor- joining method. Bar, 1 substitution per 10 nucleotides. Table 3 Nucleotide similarity values (%) and the number of nucleotide differences between Salmonella strains based on gyrB sequence D Q 4 4 7 1 4 9 . 1 S . T y p h i O (B L K Y o g y ak ar ta ) S . T y p h i R S K 5 .1 S S A S . T y p h i B P E 1 2 2 .4 C C A R * S . T y p h i B P E 1 2 7 .1 M C R * S . T y p h i N C T C 7 8 6 A Y 9 9 6 8 6 7 . 1 A Y 3 7 0 8 6 4 . 1 E F 0 6 4 8 5 3 .1 DQ447149.1 --- 16/296 13/267 20/301 17/287 16/284 233/762 278/492 420/685 S. Typhi O (BLK Yogyakarta) 94.59 --- 0/267 1/296 1/283 3/284 68/296 150/288 150/288 S. Typhi RSK 5.1 SSA 95.13 100.00 --- 0/267 0/267 2/267 59/267 131/258 131/258 S. Typhi BPE 122.4 CCA R* 93.36 99.66 100.00 --- 1/287 3/284 71/301 152/292 152/292 S. Typhi BPE 127.1 MC R* 94.08 99.65 100.00 99.65 --- 2/284 66/287 145/278 145/278 S. Typhi NCTC 786 94.37 98.94 99.25 98.94 99.30 --- 65/284 145/277 145/277 AY996867.1 69.42 77.03 77.90 76.41 77.00 77.11 --- 297/492 765/1156 AY370864.1 43.50 47.92 49.22 47.95 47.84 47.65 39.63 --- 0/506 EF064853.1 38.69 47.92 49.22 47.95 47.84 47.65 33.82 100.00 --- Samonella Typhi RSK 5.1 SSA Samonella Typhi NCTC 786 Samonella Typhi BPE 127.1 MC R* Samonella Typhi BPE 122.1 CCA Samonella Typhi BPE 122.4 CCA R* Samonella Typhi O (BLK Yogyakarta) SalmonellaTyphi strain 1474 (FJ222661.1) Klebsiella pneumoniae subsp. Rhinoscleromatis (AF303654.1) Escherichia coli (EU561348.1) Escherichia coli GJ0706-186 (EU513000.1) 83 36 100 99 0.1 Fig 3 Phylogenetic tree of Salmonella strains based on parC sequences analysis. The tree was constructed by neighbor- joining method. Bar, 1 substitution per 10 nucleotides. 4 AMARANTINI Microbiol Indones strains were nearly equal ranging from 98.94 - 100% (Table 3). Figure 3 shows the phylogenetic structure of Salmonella based on parC gene sequences. The phylogenetic analysis determined by parC sequence give rise to a tree structure which clustered all the test strains into a cluster, with control strain S. Typhi O BLK separated into different cluster as a sole species. This finding is supported by the nucleotide similarity index value above 99.0% (Table 4). An almost similar phylogenetic tree structure (Fig 4) was revealed when we analyzed the isolates based on parE gene sequences. All the tested isolates are separated into a cluster which also included some member of Enterobacter. However, a lower nucleotide similarity index was observed compared to the one produced which is based on parC gene sequences (Table 5). A lower bootstrap value was also observed, emphasizing the consistency of isolates separation based on parC gene. Table 4 Nucleotide similarity values (%) and the number of nucleotide differences between Salmonella strains based on parC sequence S a m o n el la T y p h i R S K 5 .1 S S A S a m o n el la T y p h i B P E 1 2 2 .1 C C A S a m o n el la T y p h i N C T C 7 8 6 S a m o n el la T y p h i B P E 1 2 2 .4 C C A R * S a m o n el la T y p h i B P E 1 2 7 .1 M C R * S a m o n el la T y p h i O ( B L K Y o g y ak ar ta ) S a lm o n el la T y p h i st ra in 1 4 7 4 (F J2 2 2 6 6 1 .1 ) SamonellaTyphi RSK 5.1 SSA --- 0/334 0/331 0/331 3/334 49/330 133/272 SamonellaTyphi BPE 122.1 CCA 100.00 --- 0/332 0/332 3/335 48/330 132/271 SamonellaTyphi NCTC 786 100.00 100.00 --- 0/337 1/337 47/328 132/270 SamonellaTyphi BPE 122.4 CCA R* 100.00 100.00 100.00 --- 0/336 47/328 132/270 SamonellaTyphi BPE 127.1 MC R* 99.10 99.10 99.70 100.00 --- 50/333 135/274 SamonellaTyphi O (BLK Yogyakarta) 85.15 85.45 85.67 85.67 84.98 --- 142/279 SalmonellaTyphi strain 1474 (FJ222661.1) 51.10 51.29 51.11 51.11 50.73 49.10 --- Samonella Typhi BPE 127.1 MC R* Samonella Typhi BPE 122.1 CCA Samonella Typhi RSK 5.1 SSA Samonella Typhi O (BLK Yogyakarta) Salmonella Typhi (AB072701.2) Salmonella Typhi (AM283478.1) Samonella Typhi BPE 122.4 CCA R* Samonella Typhi NCTC 786 Escherichia coli strain 90(JN565721.1) Streptococcus pneumoniae strain 5305(AY157686.1) Salmonella Typhi strain 35 (HQ698252.1) 77 96 48 58 0.1 Fig 4 Phylogenetic tree of Salmonella strains based on parE sequences analysis. The tree was constructed by neighbor- joining method. Bar, 1 substitution per 10 nucleotides. Volume 9, 2015 Microbiol Indones 5 DISCUSSION DNA sequence analysis has become increasingly popular in determining the evolutionary relationships of bacteria(Tajbakhsh et al. 2011). In the present study we therefore determined the phylogenetic structure for six strains belonging to S. Typhi based on partial gyrA and gyrB sequences. The test strains comprised of two groups, resistant and susceptible to nalidixic acid. Results of phylogenetic structure showed that all of the test strains were sharply separated, but the topology of the tree based on gyrA gene was very different to that of the gyrB. The phylgenetic tree based on gyrA gene showed that the resistant strains were organized into different clusters. Our data indicated that the gyrA nucleotide sequences showed much higher variations than gyrB. The maximum and minimum nucleotide similarityamong S. Typhi strains based on gyrA sequence was 93.35% and 73.25%, respectively (Table 2). The phylogenetic tree based on gyrB gene grouped all six S. Typhi strains togetherin a single cluster (Fig 2). Data showed that the nucleotide sequences from these groups were very similar (Table 3) ranged from 98.94 - 100%.These factsindicated that they exhibited the closest relationship. Souza et al. (2011) observed that non-fluorine quinolones such as nalidixic acid may be sufficient to generate mutations that alter the susceptibility of Salmonellaspp to fluoroquinolones. Mutations have rarely been reported in the gyrB gene (Ling et al. 2003). So, it is clear that the higher genetic variation in term of nucleotide similarity of gryA in the test strains may be mainly due to mutations. In general, the phylogenetic tree based on gyrA and gyrB genes showed the separation isolates into several clades with a better separation compared to the one based on 16S rRNA gene from the previous research (Amarantini and Budiarso 2013). The major difference is the position of the resistant isolates (BPE 122.4 CCA * * R and BPE 127.1 MC R ) which clustered with the sensitive isolates in their analysis (Fig 5). The similarity values of 16S rRNA were higher than the gyrA and gyrB sequences (Table 6). They showed the closest 16S rRNA relatedness values ( 99.42 % ≥ similarity) among all of the test strains. Comparison of gyrA and gyrB sequences and 16S r R N A s e q u e n c e s f o r p h y l o g e n e t i c a n a l y s i s demostrated that taxonomy based on 16S rRNA typing methods may not be enough for the delineation phylogenetic differences at the spesies level. The 16S Table 5 Nucleotide similarity values (%) and the number of nucleotide differences between Salmonella strains based on parE sequence S a m o n el la T y p h i R S K 5 .1 S S A S a m o n el la T y p h i B P E 1 2 7 .1 M C R * S a m o n el la T y p h i N C T C 7 8 6 S a m o n el la T y p h i B P E 1 2 2 .4 C C A R * S a m o n el la T y p h i B P E 1 2 2 .1 C C A S a m o n el la T y p h i O (B L K Y o g y ak ar ta ) S a lm o n el la T y p h i (A M 2 8 3 4 7 8 .1 ) S a lm o n el la T y p h i (A B 0 7 2 7 0 1 .2 ) S a lm o n el la T y p h i st ra in 3 5 (H Q 6 9 8 2 5 2 .1 ) SamonellaTyphi RSK 5.1 SSA --- 0/198 4/199 4/196 0/197 2/197 1/199 1/199 107/193 SamonellaTyphi BPE 127.1 MC R* 100.00 --- 4/198 4/196 0/197 2/197 1/198 1/198 107/192 SamonellaTyphi NCTC 786 97.99 97.98 --- 0/200 4/197 6/200 4/207 4/207 113/203 SamonellaTyphi BPE 122.4 CCA R* 97.96 97.96 100.00 --- 4/196 6/196 3/198 3/198 105/193 SamonellaTyphi BPE 122.1 CCA 100.00 100.00 97.97 97.96 --- 2/196 1/197 1/197 106/191 SamonellaTyphi O (BLK Yogyakarta) 98.98 98.98 97.00 96.94 98.98 --- 3/200 3/200 108/194 Salmonella Typhi (AM283478.1) 99.50 99.49 98.07 98.48 99.49 98.50 --- 1/481 134/233 Salmonella Typhi (AB072701.2) 99.50 99.49 98.07 98.48 99.49 98.50 99.79 --- 134/233 Salmonella Typhi strain 35 (HQ698252.1) 44.56 44.27 44.33 45.60 44.50 44.33 42.49 42.49 --- 6 AMARANTINI Microbiol Indones Samonella Typhi BPE 127.1 MC R* Samonella Typhi BPE 122.4 CCA R* Samonella Typhi BPE 122.1 CCA Samonella Typhi NCTC 786 Samonella Typhi ATCC 19430T ( Z47544) Samonella Typhi RSK 5.1 SSA Samonellabongori (AF029227) Escherichia coli ATCC 25922 (X80724.1) 65 100 0.005 Fig 5 Phylogenetic tree of Salmonella strains based on 16S rRNA sequences analysis. The tree was constructed by neighbor- joining method. Bar, 5 substitution per 5000 nucleotides. Table 6 Nucleotide similarity values (%) and the number of nucleotide differences between Salmonella strains based on 16S rRNA S . T y p h i B P E 1 2 2 .4 C C A ( R * ) S . T y p h i B P E 1 2 7 .1 M C ( R * ) S . T y p h i B P E 1 2 2 .1 S . T y p h i R S K 5 .1 S S A S . T y p h i N C T C 7 8 6 Z 4 7 5 4 4 A F 0 2 9 2 2 7 X 8 0 7 2 4 .1 S. Typhi BPE 122.4 CCA (R*) --- 3/1383 0/1381 2/1382 3/1381 5/1381 33/1377 45/1374 S. Typhi BPE 127.1 MC (R*) 99.78 --- 3/1381 5/1382 6/1381 8/1381 36/1377 48/1374 S. Typhi BPE 122.1 100.00 99.78 --- 2/1381 3/1381 5/1381 33/1377 45/1374 S. Typhi RSK 5.1 SSA 99.86 99.64 99.86 --- 3/1381 5/1381 33/1377 45/1374 S. Typhi NCTC 786 99.78 99.57 99.78 99.78 --- 6/1381 34/1377 46/1374 Z47544 99.64 99.42 99.64 99.64 99.57 --- 36/1497 48/1446 AF029227 97.60 97.39 97.60 97.60 97.53 97.60 --- 39/1442 X80724.1 96.72 96.51 96.72 96.72 96.65 96.68 97.30 --- rRNA genes couldn't be used as a suitable marker for classification of closely related bacterial spesies (Tajbakhsh et al. 2011). It was useful only for describing phylogenetic relationships between distantly related Enterobacteriaceae and can not be applied for intrageneric relationship (Dauga 2002). In the present study, we also noted that the a p p l i c a t i o n o f g y r A t y p i n g s h o w e d a b e t t e r classification than gyrB. The gyrA gene exhibited high variation in nucleotide sequences (73.25 - 93.35% similarity). Because of this, the gyrA gene provided higher resolution than the gyrB gene. It is, therefore encouraging that gyrA was found to be the best marker for classification. Based on these results, it is clear there are nucleotide polymorphisms occur among gyrA, gyrB, parC, and parE which result in defined clustering of the tested isolates. Nonetheless, it is recommended to employ parC as a preferred gene to distinguish Salmonella and its close relative, like member of Enterobacter, as it is sensitive and specific. A further study need to be done to ensure this assumption as full sequence of each QRDR genes were not included in this study. ACKNOWLEDGMENTS This research was funded by the Directorate General of Higher Education, Department of National Education (Hibah Fundamental) 2014, contract no:1348/K5/KL/2013 date 14-05-2014. REFERENCES Afzal A, Sarwar Y, Ali A, Maqbool, Salman M, Habeeb MA, Haque A. 2013. Molecular evaluation of drug resistance in clinical isolates of Salmonella enterica serovar Typhi from Pakistan. J Infect Dev Ctries. 7(12): 929-940. doi: 10.3855/jidc.3154. Amarantini C, Budiarso TY. 2013. 16S rDNA typing of Volume 9, 2015 Microbiol Indones 7 Salmonellatyphi strains from different geographical locations in Sumba island East Nusa Tenggara 8 AMARANTINI Microbiol Indones Page 1 Page 2 Page 3 Page 4 Page 5 Page 6 Page 7 Page 8