J. Microbiol. Biotechnol. (2014), 24(8), 1026–1033 http://dx.doi.org/10.4014/jmb.1310.10043 Research Article Review jmb Senior Thai Fecal Microbiota Comparison Between Vegetarians and Non-Vegetarians Using PCR-DGGE and Real-Time PCR Supatjaree Ruengsomwong1,2,3, Yuki Korenori4, Naoshige Sakamoto4, Bhusita Wannissorn3, Jiro Nakayama4, and Sunee Nitisinprasert1,2* 1Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Chatuchak, Bangkok 10900, Thailand 2Center for Advanced Studies for Agriculture and Food, Kasetsart University Institute for Advanced Studies, Kasetsart University (CASAF, NRU-KU), Chatuchak, Bangkok 10900, Thailand 3Bioscience Department, Thailand Institute of Scientific and Technological Research, Technopolis, Pathum Thani 12120, Thailand 4Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 812-8581, Japan Received: October 14, 2013 Revised: April 2, 2014 The fecal microbiotas were investigated in 13 healthy Thai subjects using polymerase chain Accepted: April 14, 2014 reaction denaturing gradient gel electrophoresis (PCR-DGGE). Among the 186 DNA bands detected on the polyacrylamide gel, 37 bands were identified as representing 11 species: Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides uniformis, Bacteroides vulgatus, First published online Clostridium colicanis, Eubacterium eligenes, E. rectale, Faecalibacterium prausnitzii, Megamonas April 18, 2014 funiformis, Prevotella copri, and Roseburia intestinalis, belonging mainly to the groups of *Corresponding author Bacteroides, Prevotella, Clostridium, and F. prausnitzii. A dendrogram of the PCR-DGGE divided Phone: +66-2562-5089; the subjects; vegetarians and non-vegetarians. The fecal microbiotas were also analyzed using Fax: +66-2579-4096; a quantitative real-time PCR focused on Bacteroides, Bifidobacterium, Enterobacteriaceae, E-mail: [email protected] Clostrium coccoides-Eubacterium rectale, C. leptum, Lactobacillus, and Prevotella. The non- vegetarian and vegetarian subjects were found to have significant differences in the high abundance of the Bacteroides and Prevotella genera, respectively. No significant differences were found in the counts of Bifidabacterium, Enterobacteriaceae, C. coccoides-E. rectale group, C. leptum group, and Lactobacillus. Therefore, these findings on the microbiota of healthy Thais consuming different diets could provide helpful data for predicting the health of South East pISSN 1017-7825, eISSN 1738-8872 Asians with similar diets. Copyright© 2014 by The Korean Society for Microbiology Keywords: Fecal microbiota, PCR-DGGE, quantitative real-time PCR, Bacteroides, Prevotella and Biotechnology Introduction rapidity, and accuracy [6, 9, 27, 28, 31]. The most widely used molecular methods are based on the 16S rRNA The gastrointestinal microbiota of humans represent an sequence, such as fluorescent in situ hybridization (FISH), enormous number of 1011-1014 bacterial cells with terminal restriction fragment length polymorphism (T- approximately 500-1,000 different species [9, 31]. The RFLP), denaturing/temperature gradient gel electrophoresis techniques generally used for studying human microbiotas (DGGE/TGGE), and pyrosequencing. Although DGGE is an can be broadly divided into cultivation and molecular effective and popular fingerprinting technique for separating methods. However, culture-dependent methods are gradually bacterial communities in environmental systems, a disappearing, as they are labor- and time-consuming, quantitative real-time PCR is used to quantify interesting painstaking as regards detail, incomplete in terms of data, gut microbiota [18, 19]. The variation of the microbiota and have a community bias, whereas molecular techniques development in the human gastrointestinal tract (GI tract) are becoming more popular owing to their broad coverage, is influenced by many factors, including the composition of August 2014 ⎪ Vol. 24⎪ No. 8 1027 Ruengsomwong et al. the gut, the person’s age, and the consumption style or diet 0.1 mm zirconium beads from Bio Spec Products (OK, USA) by [4, 10, 13, 14, 23, 33, 34]. Owing to growing health concerns, vortexing at 2,000 rpm for 1 min, followed by keeping on ice for 1 Thai people are now tending to eat more healthy food, like min. This step was repeated three times. Thereafter, the total fruit and vegetables. Vegetables are known as a low fat bacterial genomic DNA was extracted according to the Qiagen kit source containing good amounts of vitamins and minerals. instructions. The DNA was eluted with sterilized pure water and kept at -20oC until use. Various publications have also showed that vegetarians or vegans have a lower risk of cancer than meat eaters [7, 24]. PCR-DGGE of 16S rRNA Thus, different consumption behaviors may result in a To access the PCR-DGGE, two primers, namely HDA1-GC and different microbial community and provide key bacterial HDA2 [30], were used to amplify the 16S rRNA gene of each species that maintain good health. To date, most research sample. The sequences for the primers were as follows: HDA1- has focused on the gut microbiota of Asian adults and GC, 5’-CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG elders [13, 14, 25], Asian vegetarians [12, 16], vegetarians in GCA CGG GGG GAC TCC TAC GGG AGG CAG CAG T-3’, and Europe [17, 22], predominantly vegetarian children in HDA2, 5’-GTA TTA CCG CGG CTG CTG GCA C-3’. The PCR- Africa [8], and high-carbohydrate-consuming Americans DGGE conditions were as follows: 94°C for 5 min, 30 cycles at [33]. However, there has been no report on the gut 94°C for 40 sec, 58°C for 20 sec, and 72°C for 1 min, and a final microbiota of Thai vegetarians and non-vegetarians. In a elongation at 72°C for 5 min [30]. The expected PCR product size report from the National Cancer Institute (NCI, Thailand), was 250 bp. The PCR-DGGE profile was performed using a Dcode System apparatus from BioRad (CA, USA). The electrophoresis was between 2007 and 2011, colorectal cancer was among the run using a 25-65% denaturing gradient with 100% corresponding top three cancers found in males and females [2]. to 7 M urea and 40% formamide in an 8% polyacrylamide gel, at Consequently, this study was interested in the microbiota 80 volts for 1 h and subsequently at 100 volts for 6 h at 60°C. of Thais who are vegetarians and Thais who are meat After the PCR-DGGE running step, a dendrogram was constructed eaters. Two methods, PCR-DGGE and real-time PCR, were to cluster the PCR-DGGE profile. The similarities between the used for a comparative analysis of the gut microbiota from PCR-DGGE profiles were analyzed based on the locations of the each group. DNA bands on the PCR-DGGE gel using SYNGENE Gene Tools ver. 4.03(b) and SYNGENE Gene Directory Application ver. 2.01 (c) Materials and Methods from SYNGENE, a division of Synoptic Ltd., Dice’s similarity coefficient, and the unweighted pair group method with arithmetic Fecal Samples mean (UPGMA). Six and seven fecal samples were obtained from healthy non- To determine the bacterial species, the bands of interest were vegetarians and healthy vegetarians, respectively. All the subjects cut and eluted in sterile pure water. Each eluted band was then re- had regular bowel habits, including no change of defecation amplified with the HDA1-GC and HDA2 primers and run on the frequency, no history of gastrointestinal disease, such as gastritis, DGGE system at a suitable gradient concentration to check the peptic ulcers, gastric cancer, colorectal cancer, or inflammatory purity of the cut band. Following the purity check, each band was bowel disease (IBD), no diarrhea in the month preceding the re-amplified without a GC clamp and then purified using a sampling, and no family history of colorectal cancer. None of the QIAquick PCR Purification kit (Qiagen). The purified PCR products subjects had received any antibiotic treatment within at least one were analyzed using a direct sequencing analysis performed by month prior to this study. The vegetarian volunteers were ovo- 1st BASE, Malaysia. The fragment of interest was identified using lacto vegetarians or lacto-vegetarians and had been vegetarians BLAST in the NCBI and Eztaxon databases. for at least 3 years before participating in this study. A stool sampling kit consisting of a sample collection tube, cotton swabs, Real-time PCR Analysis of Gut Microbiota and sterile tissue paper together with a questionnaire about each Seven bacterial groups were quantified in each subject using a individual’s consumption behavior and consent form were given quantitative real-time PCR (LightCycler 480; Roche, The Netherlands) to each subject. The study protocol and consent documents were based on previously published specific primers for Prevotella, approved by the Institute for the Development of Human Bacteroides (based on the Bacteroides fragilis group), Clostridium Research Protections (IHRP) under ethic approval No. IHRP 311. leptum group [20], Bifidobacterium, Clostridium coccoides-Eubacterium rectale group [29], Enterobacteriaceae [3], and Lactobacillus [11]. DNA Extraction Genomic DNA (50-100 ng) from each subject was used as the The total bacterial genomic DNA from each sample was template in a reaction volume of 20 µl. Genomic DNA extracted extracted using zirconium beads and a QIAamp Stool Mini Kit from Prevotella nigrescenes JCM 12250T, Bacteroides fragilis ATCC from Qiagen (Hilden, Germany). In brief, each