1.MI731-Eveline Ayu NEW Available online at http://jurnal.permi.or.id/index.php/mionline DOI: 10.5454/mi.8.1.2ISSN 1978-3477, eISSN 2087-8575 Vol 8, No 1, Maret 2014, p 9-15 *Corresponding author; Phone/Fax: +62-21-5703306/5727615 ext. 450/+62-21-5719060, Email: antoniussuwanto@gmail.com Tempeh is an indigenous Indonesian fermented food and has become an important part of the Indonesian diet for hundreds years. Tempeh is consumed in relatively large portion and can be found in a variety of types of cooking and processing methods. Excellent protein quality has made tempeh become a meat substitute and becomes popular among vegetarians (Liem et al.1977). The process of tempeh making in Indonesia is still using conventional methods with uncontrolled condition (Barus et al. 2008). During the fermentation process, not only fungi involved but also bacteria play important roles in the formation of flavor and nutrition. Bacterial growth during tempeh production begins in the process of soybean soaking. During the fermentation process, the producers inadvertently adding bacteria so that the bacteria eventually become an inseparable part of tempeh, and even has an important role in determining the quality of tempeh (Barus et al. 2008; Seumahu et al. 2013). Several studies have reported the presence of bacteria in tempeh as Klebsiella pneumoniae and Citrobacter freundii (Keuth and Bisping 1994), and also bacteria of the phylum Proteobacteria and Firmicutes (Seumahu et al. 2012) which are reported as bacteria producing vitamin B in tempeh. Other 12 contaminants such as Brevibacterium epidermidis and Micrococcus luteus known to play a role in the formation of antioxidants in tempeh (Klus and Barz 1995). Furthermore, bacteria in tempeh is also known as one of the factors that play a role in the formation of a bitter taste in tempeh (Barus et al. 2008). A diverse array of lactid acid bacteria and yeasts also play important role in Indonesian tempeh production (Efriwati et al. 2013). K. pneumoniae which is included in family Enterobacteriaceae is also known both of causing Tempeh is important traditional Indonesian fermented food made​​ from soybeans employing Rhizopus oligosporus or R. microsporus. During the process of tempeh production, some bacteria from the environment and tempeh starter become an integral part of tempeh, and even have important roles in determining the final quality of tempeh it self. Several studies reported the presence of Klebsiella pneumoniae in tempeh as one of vitamin B12 producing bacteria in tempeh. However, K. pneumoniae also known as opportunistic pathogens causing pneumonia and liver abscess in human. In this study, Enterobacterial Repetitive Intergenic Consensus- Polymerase Chain Reaction (ERIC-PCR) was employed to determine genetic diversity of K. pneumoniae isolated from tempeh and compared them with medical isolates. The result indicated that isolates from tempeh were genetically distinct from those of medical isolates. Key words: ERIC-PCR, tempeh Tempe merupakan salah satu makanan utama tradisional Indonesia yang terbuat dari kacang kedelai dengan fermentasi menggunakan cendawan Rhizopus oligosporus atau R. microsporus. Selama proses pengolahan, bakteri yang berasal dari lingkungan dan inokulum awal menjadi bagian yang tidak terpisahkan dari tempe, bahkan memiliki peranan yang penting dalam menentukan kualitas akhir pada tempe. Beberapa penelitian telah melaporkan keberadaan Klebsiella pneumoniae pada tempe sebagai salah satu bakteri penghasil vitamin B12 pada tempe. Akan tetapi, K. pneumoniae juga dikenal sebagai patogen oportunis penyebab penyakit pneumonia dan abses hati pada manusia. Pada penelitian ini, metode Enterobacterial Repetitive Intergenic Consensus- Polymerase Chain Reaction (ERIC-PCR) digunakan untuk membandingkan keragaman genetik K. pneumoniae pada tempe dibandingkan dengan isolat medis. Hasil penelitian ini menunjukkan bahwa secara genetik pneumoniae pada tempe berbeda dengan isolat medis. Kata kunci: ERIC-PCR, tempe Klebsiella pneumoniae, Klebsiella pneumoniae, Klebsiella pneumoniae from Indonesian Tempeh were Genetically Different from that of Pathogenic Isolates 1 1,2 1 EVELINE AYU , ANTONIUS SUWANTO *, AND TATI BARUS 1 Department of Biology, Faculty of Biotechnology, Universitas Katolik Atma Jaya, Jalan Jenderal Sudirman 51, Jakarta 12930, Indonesia; 2 Department of Biology, Faculty of Mathematics and Natural Sciences, Institut Pertanian Bogor, Darmaga Campus, Bogor 16680, Indonesia pneumonia disease, an acute infection that attacks the alveoli (Gori et al. 1996) and liver abscess in human (Wang et al. 1998). Therefore, the study of genetic diversity is important for the identification and characterization of bacterial pathogenicity (Rademaker and de Bruijn 1997). One of the many molecular techniques for the study of genetic diversity is Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR. ERIC sequences are short sequences, which is 126 bp long, with sequences that are conserved as internal repeat and as non-coding sequences (Lupski et al. 1992). This technique is often used because it is simple, rapid, reproducible, and discriminative (Olive et al. 1999) and has been successfully analyzed the diversity of different types of bacteria, such as Mycobacterium tuberculosis (Sechi et al. 1998) and Vibrio parahaemolyticus (Khan et al. 2002). ERIC- PCR has also been used to analyzed genetic diversity of Klebsiella spp. isolated from tempeh (Barus et al. 2013), as well as to study genetic heterogeneity of many types of Vibrio cholerae (Waturangi et al. 2012). MATERIALS AND METHODS This research was conducted in Research Laboratory, Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia from May to December 2013. Medical isolates of K. pneumoniae. Four medical isolates ware used for comparison with K. pneumoniae from tempeh, i.e. K. pneumoniae ATCC BAA-2146, K. pneumoniae subsp. pneumoniae ATCC 10031, K. pneumoniae ATCC 35657, and one K. pneumoniae isolate originated from pneumonia patient, named as FK isolate, collection of Department of Microbiology, Faculty of Medicine, Atma Jaya Catholic University. K. pneumoniae isolates from Tempeh. Tempeh EMP and WJB was produced in Bogor, West Java, Indonesia (Barus et al. 2008). A 10 g of fresh tempeh was placed into 90 mL of sterile physiological salt 0.85% (w/v) NaCl and homogenized in orbitar shaker (Yih Der) at a speed of 24 x g for one min. Dilution was -1 -6 made ​​from 10 until 10 and a 100 µL from dilution of -4 -5 -6 10 , 10 , and 10 was spread on Eosin Methylene Blue (EMB) Agar (Oxoid) and incubated overnight at 37 °C. A single purple mucoid colony was typical character of K. pneumoniae. These colonies were further analyzed by cultivating them on Simmons’ Citrate Agar (SCA) (Difco), and incubated at 37 °C for 24 h. Tempeh sampling were conducted twice, i.e. August and October 2013. Klebsiella sp. 135 isolated from tempeh 10 AYU ET AL. Microbiol Indones was used for control (Maysella 2010). Analysis of 16S rRNA Genes. Suspected blue colonies on Simmons’ Citrate Agar were further verified using sequencing of genes encoding 16S rRNA. The whole cells from single colonies on plates were used directly in PCR reaction as described by Rademaker and de Bruijn in 1997. The 16S rRNA gene was amplified employing a PCR machine (Applied Biosystems, 2720 Thermal Cycler) using primer 63f (5’- CAGGCCTAACACATGCAAGTC- 3’) and 1387r (5’- GGGCGGWT GTACAAGGC -3’) (Marchesi et al. 1998). Total PCR volume was 50 µl containing 2 µL DNA template, 25 µL GoTaq Green® Master Mix (Promega), 2 µL primer forward dan -1 reverse (25 pmol µL ) dan 19 µL nuclease free water. The PCR protocol was as follows: initial denaturation at 94 °C for 5 min, denaturation at 92 °C for 30 s, annealing at 62 °C for 30 s, elongation at 72 °C for 30 s, and post extention at 72 °C for 7 min. The cycle was repeated for 30 times. A 5 µL of PCR amplification products were further verified by electrophoresis in 1% agarose (Bioline) in 1x TAE buffer for 60 min, 80 V. Sequencing of PCR products were performed in Macrogen Inc., Korea, and were analyzed employing a program SeqTrace. Sequencing results were compared to the database with the Basic Local Alignment Search Tool (BLAST) program which is provided by National Centre of Biotechnology Information (NCBI). Genetic Profiling of K. pneumoniae Isolates. 13 bacterial isolates were identified as K. pneumoniae from EMP tempeh and 10 isolates from WJB tempeh. A total PCR volume used was 25 µL containing 12.5 µL GoTaq Green® Master Mix (Promega), 1 µL of 25 pmol ERIC1R (5’-ATGTAAGCTCCTGGGGATTCA C-3’), 1 µL of 25 pmol ERIC2F (5’-AAGTAAGTGAC TGGGGTGAGCG-3’)Give references for the primers (Versalovic et al. 1991), 9.5 µL nuclease free water, and 1 µL DNA template which was obtained directly from the isolates using a sterile toothpick. The PCR protocol was as follows: initial denaturation at 95 °C for 7 min, denaturation at 95 °C for 30 s, annealing at 49 °C for 1 min, elongation at 65 °C for 8 min, and post extention at 65 °C for 16 min (Applied Biosystems, 2720 Thermal Cycler). The PCR cycle was used 30 times. A 5 µL of PCR products was verified by electrophoresis for 90 min and 70 V, on 1% agarose in 1x TAE buffer. Formed band profiles were observed under the UV transilluminator. Formed band profiles were then compared as biner number and analyzed using FreeTree (Hampl et al. 2001) and TreeView to construct a phylogenetics tree (http://taxonomy.zoology.gla.ac.uk/ rod/treeview.html. RESULTS Isolation of K. pneumoniae. A total of 58 bacterial isolates (Table 1) were isolated from EMP and WJB tempeh. The bacterial colonies were purple in the center of colony, mucoid, and rounded shape on EMB medium. The colonies of these isolates changed from green to blue on Simmons’Citrate medium, which is specific character of Klebsiella sp. Fig 1A and 1B showed hypermucoviscocity of K. pneumoniae ATCC35657 or FK colonies when touched with inoculating loop. However, colonies of K. pneumoniae from EMP (Fig 1C) and WJB tempeh (Fig 1D) did not show hypermucoviscosity. This character showed significant phenotypic difference between tempeh and those of medical isolates. Analysis of 16S rRNA Genes. A total of 18 isolates from the EMP tempeh and 25 isolates from WJB tempeh were selected for further amplification of genes encoding 16S rRNA. Based on 16S rRNA gene sequence alignments with the NCBI database, K. pneumoniae were identified in 13 isolates obtained from EMP tempeh and 10 isolates from WJB tempeh (Table 1). The other microorganisms were found from EMP tempeh based on 16S alignment analysis were: Klebsiella sp., Rhizobium sp., and Enterobacter sp. while Bacterium sp., Klebsiella sp., and Cronobacter sakazakii were isolated from WJB tempeh. Genetic Profiling of K. pneumoniae Isolates. A total 23 isolates which were identified as K. pneumoniae were further subjected to ERIC-PCR analysis in order to compare their genetic diversity from those of medical isolates. ERIC-PCR of 13 isolates of K. pneumoniae from EMP tempeh showed a similar pattern (Fig 2). The ERIC-PCR profiles of medical isolates showed a diverse patterns but distinctively different from tempeh isolates. This result indicated that K. pneumoniae presence in tempeh was not the same as pathogenic K. pneumoniae. Similar result were also found when we analysed isolates from WJB tempeh (Fig 3) which showed different ERIC- PCR profiles from the medical isolates. Although genetic profiles of K. pneumoniae isolates in WJB tempeh were more varied than EMP tempeh, we found noi dentical profiles when compared to those of medical isolates. The resulting electrophoresis band profiles were further converted into binary data matrix. The results were used to construct a phylogenetic tree using FreeTree program and TreeView. Fig 4 showed the genetic relationship of K. pneumoniae isolated from EMP tempeh. Based on this analysis, the medical isolates clustered into a separate and distict group. Tempeh isolates formed two groups, the first one contains isolates which have similar ERIC profiles, i.e., I EMP16, I EMP13, I EMP9, I EMP8, I EMP5, I EMP4, and I EMP. The other group contains of I EMP1, II EMP5, II EMP2, II EMP4, II EMP1, and II EMP3. Isolates from WJB tempeh formed a separate group under one branch except one isolate, I WJB2 (Fig 5). Again, in this analysis, the medical isolates formed a separate group outside the branches which contained isolates from WJB. DISCUSSION K. pneumoniae presence in both of tempeh samples (EMP and WJB) and consistantly exist from the first Volume 8, 2014 Microbiol Indones 11 Tempeh sample Number of sampling Number of Isolates Sequencing of 16S rRNA EMB agar SCA DNA Sequence Alignment (K. pneumoniae) EMP I 18 18 10 8 II 8 8 8 5 Total of EMP 26 26 18 13 WJB I 17 10 10 7 II 15 15 15 3 Total of WJB 32 25 25 10 Grand Total 58 51 43 23 Table 1 Klebsiela pneumoniae from fresh tempeh Microbiol Indones12 AYU ET AL. and second sampling of each of these tempeh samples. The presence of K. pneumoniae in tempeh have been reported before (Keuth et al. 1994, Barus et al. 2008). Growth of K. pneumoniae on EMB medium showed distinctive colony morphology, which were dark purple in the center, mucoid, and rounded shape. C D A B Fig 1 The phenotype of K. pneumoniae ATCC 35657 (A), FK isolate (B), K. pneumoniae from EMP (C), and WJB tempeh (D). 700 bp Fig 2 ERIC-PCR from K. pneumoniae from EMP tempeh, ie. (M) Molecular markers, (A) ATCC 10031, (B) ATCC 2014, (C) ATCC 35657, (D) FK, (E) Klebsiella sp. 135, (F) I EMP1, (G) I EMP3, (H) I EMP4, (I) I EMP5, (J) I EMP8, (K) I EMP9, (L) I EMP13, (N) I EMP16, (O) II EMP1, (P) II EMP2, (Q) II EMP3, (R) II EMP4, and (S) II EMP5. Volume 8, 2014 Microbiol Indones 13 700 bp Fig 3 ERIC-PCR profiles of K. pneumoniae from WJB tempeh, ie. (M) Molecular markers, (A) ATCC 10031, (B) ATCC 2014, (C) ATCC 35657, (D) FK, (E) Klebsiella sp. 135, (F) I WJB1, (G) I WJB2, (H) I WJB3, (I) I WJB4, (J) I WJB5, (K) I WJB6, (L) I WJB16, (N) II WJB3, (O) II WJB5, and (P) II WJB8. Medical isolate Medical isolates Group 2 Group 3 Medical isolates Group 2 Group 3 Medical isolate Fig 5 Philogenetic tree generated from ERIC-PCR profiles of K. pneumoniae isolated from WJB tempeh. Fig 4 Philogenetic tree generated from ERIC-PCR profiles of K. pneumoniae isolated from EMP tempeh. disease associated with K. pneumoniae in tempeh. On the other hand, their presence in tempeh might be beneficial due to their ability to synthesize vitamin B12 and their immunomodulatory effects in human. To conlude tempeh produced in Indonesia naturally harbors K. pneumoniae with unique genomic profiles. K. pneumoniae from tempeh samples in this study showed that they were genetically different from isolates known to be pathogenic to human. ACKNOWLEDGMENT This work was supported by DANA DIPA IPB (code : 2013.089.521219). We also acknowlegde the Department of Microbiology, Faculty of Medicine, Universitas Katolik Atma Jaya for the FK isolate. 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Mutation in magA is one of the factors that mutants lost the hypermucoviscosity phenotype and became susceptible tophagocytes and a virulent to mice (Fang et al. 2004). 16S rRNA gene sequences are the most common house keeping genetic marker used for identifying bacteria in the laboratory (Janda et al. 2007). Although 16S rRNA gene (approximately 1 500 bp ) is large enough for bioinformatics purposes, but usually it is not reliable for intra-strain differentiation. For example, Escherichia coli O104: H4 that is known pathogenic as well as isolate in recent out breaking Germany (Mellmann et al. 2011) were confirmed only as E. coli based on 16S DNA sequence analysis. We confirmed colonies obtained from tempeh samples as K. penumoniae based on 16S DNA sequence analysis before subjected them for ERIC- PCR analysis to reveal intra-species genetic diversity (Barus et al. 2013). In this study, ERIC-PCR method has been successfully employed to differentiate genetic profiles of K. pneumoniae isolated from tempeh and those of medical isolates. The ERIC-PCR profiles of K. pneumoniae from EMP tempeh (Fig2) and WJB tempeh (Fig3) were different from those of medical isolates. Keuth and Bispingin 1994 reported that K. pneumoniae isolated from Indonesian tempeh were negative for three known enterotoxins, i.e. Shiga-like toxin SLTIIA, heat-labil enterotoxin LTIh, and heat- stable enterotoxin STIh. Therefore, intraspecies genetic diversity within K. pneumoniae might reflect different phenotypes which could make the pathogenic or non-pathogenic. The phylogenetic analysis showed two separate clusters representing K. pneumoniae from tempeh and the medical isolates. The phylogenetic trees generated from EMP tempeh showed unambiguously that tempeh isolates were genetically different from pathogenic K. pneumoniae (Fig4). 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