Senior Thai Fecal Microbiota Comparison Between Vegetarians and Non-Vegetarians Using PCR-DGGE and Real-Time PCR

Supatjaree  Ruengsomwong; Yuki  Korenori; Naoshige  Sakamoto; Bhusita  Wannissorn; Jiro  Nakayama; Sunee  Nitisinprasert

doi:10.4014/jmb.1310.10043

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Microbial Ecology and Diversity

Senior Thai Fecal Microbiota Comparison Between Vegetarians and Non-Vegetarians Using PCR-DGGE and Real-Time PCR

Supatjaree Ruengsomwong ^{1, 2, 3}, Yuki Korenori ⁴, Naoshige Sakamoto ⁴, Bhusita Wannissorn ³, Jiro Nakayama ⁴ and Sunee Nitisinprasert ^{1, 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; Accepted: April 14, 2014

J. Microbiol. Biotechnol. 2014; 24(8): 1026-1033

Published August 28, 2014 https://doi.org/10.4014/jmb.1310.10043

Abstract

The fecal microbiotas were investigated in 13 healthy Thai subjects using polymerase chain 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, Clostridium colicanis, Eubacterium eligenes, E. rectale, Faecalibacterium prausnitzii, Megamonas funiformis, Prevotella copri, and Roseburia intestinalis, belonging mainly to the groups of Bacteroides, Prevotella, Clostridium, and F. prausnitzii. A dendrogram of the PCR-DGGE divided the subjects; vegetarians and non-vegetarians. The fecal microbiotas were also analyzed using a quantitative real-time PCR focused on Bacteroides, Bifidobacterium, Enterobacteriaceae, Clostrium coccoides-Eubacterium rectale, C. leptum, Lactobacillus, and Prevotella. The nonvegetarian 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 Asians with similar diets.

Keywords

faecal microbiota, PCR-DGGE, quantitative real-time PCR, Bacteroides, Prevotella

References

Arumugam M, Raes J, Pelletier E, Paslier DL, Yamada T, Mende DR, et al. 2011. Enterotypes of the human gut microbiome. Nature 473: 174-180.
Attasara P, Buasom R. 2012. Hospital-based Cancer Registry 2011, pp 1-4. Information Technology Division, National Cancer Institute, Bangkok.
Bartosch S, Fite A, Macfarlane GT, McMurdo ME. 2004. Characterization of bacterial communities in feces from healthy elderly volunteers and hospitalized elderly patients by using real-time PCR and effects of antibiotic treatment on the fecal microbiota. Appl. Environ. Microbiol. 70: 35753581.
Benno Y, Endo K, Mizutani T, Namba Y, Komori T, Mitsuoka T. 1989. Comparison of fecal microflora of elderly persons in rural and urban areas of Japan. Appl. Environ. Microbiol. 55: 1100-1105.
Claesson MJ, Jeffery IB, Conde S, Power SE, O’Connor EM, Cusack S, et al. 2012. Gut microbiota composition correlates with diet and health in the elderly. Nature 488: 178-184.
Clarridge J. 2004. Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clin. Microbiol. Rev. 17: 840-862.
Crowe FL, Roddam AW, Key TJ, Appleby PN, Overvad K, Jakobsen MU, et al. 2011. Fruit and vegetable intake and mortality from ischaemic heart disease: results from the European Prospective Investigation into Cancer and Nutrition (EPIC)-Heart study. Eur. Heart J. 465: 1-9.
De Filippo C, Cavalieri D, Di Paola M, Ramazzotti M, Poullet JB, Massart S, et al. 2010. Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. PNAS 107: 14691-14696.
Dethlefsen L, Eckburg PB, Bik EM, Relman DA. 2006. Assembly of the human intestinal microbiota. Trends Ecol. Evol. 21: 517-523.
Favier CF, Vaughan EE, de Vos WM, Akkermans AD. 2002. Molecular monitoring of succession of bacterial communities in human neonates. Appl. Environ. Microbiol. 68: 219-226.
Haarman M, Knol J. 2006. Quantitative real-time PCR analysis of fecal Lactobacillus species in infants receiving a prebiotic infant formula. Appl. Environ. Microbiol. 72: 23592365.
Hayashi H, Sakamoto M, Benno Y. 2002. Fecal microbial diversity in a strict vegetarian as determined by molecular analysis and culture cultivation. Microbiol. Immunol. 46: 819831.
Hayashi H, Sakamoto M, Kitahara M, Benno Y. 2003. Molecular analysis of fecal microbiota in elderly individuals 16S rDNA libraries and T-RFLP. Microbiol. Immunol. 47:5557-5570.
Hayashi H, Takahashi R, Nishi T, Sakamoto M, Benno Y. 2005. Molecular analysis of jejunal, ileal, caecal and recto sigmoidal human colonic microbiota using 16S rRNA gene libraries and terminal restriction fragment length polymorphism. J. Med. Microbiol. 54: 1093-1101.
Jeffery IB, O’Toole PW. 2013. Diet-microbiota interactions and their implications for healthy living. Nutrient 5: 234-252.
Kabeerdoss J, Devi RS, Mary RR, Ramakrishna BS. 2012. Faecal microbiota composition in vegetarians: comparison with omnivores in a cohort of young women in southern India. Br. J. Nutr. 108: 953-957.
Liszt K, Zwielehner J, Handschur M, Hippe B, Thaler R, Haslberger AG. 2009. Characterization of bacteria, Clostridia and Bacteroides in feces of vegetarians using qPCR and PCRDGGE fingerprinting. Ann. Nutr. Metab. 54: 253-257.
Mariat D, Firmesse O, Levenez F, Guimarães VD, Sokol H, Doré J, et al. 2009. The Firmicutes/Bacteroidetes ratio of the human microbiota changes with age. BMC Microbiol. 9: 123.
Matsuda K, Tsuji H, Asahara T, Matsumoto K, Takada T, Nomoto K. 2009. Establishment of an analytical system for the human fecal microbiota, based on reverse transcriptionquantitative PCR targing of multicopy rRNA molecules. Appl. Environ. Microbiol. 75: 1961-1969.
Matsuki T, Watanabe K, Fujimoto J, Takada T, Tanaka R. 2004. Use of 16S rRNA gene-targeted group-specific primers for real-time PCR analysis of predominant bacteria in human feces. Appl. Environ. Microbiol. 70: 7220-7228.
Maukonen J, Mättö J, Satokari R, Söderlund H, MattilaSandholm T, Saarela M. 2006. PCR DGGE and RT-PCR DGGE show diversity and short-term temporal stability in the Clostridium cocccoides-Eubacterium rectale group in the human intestinal microbiota. FEMS Microbiol. Ecol. 58: 517528.
Matijaši B B, O bermajer T, L ipoglavšek L, G rabnar I , Gorazd A, Rogelj I. 2013. Association of dietary type with fecal microbiota in vegetarians and omnivore in Slovenia. Eur. J. Nutr. DOI: 10.1007/s00394-013-0607-6.
Moore W, Moore L. 1995. Intestinal floras of populations that have a high risk of colon cancer. Appl. Environ. Microbiol. 61: 3202-3207.
Nair P, Mayberry JF. 1994. Vegetarianism, dietary fibre and gastro-intestinal disease. Digest. Dis. 12: 177-185.
Nam YD, Jung MJ, Roh SW, Kim MS, Bae JW. 2011. Comparative analysis of Korean human gut microbiota by pyrosequencing. PLoS ONE 6: e22109.
NCBI. Prevotella copri DSM 18205 P_copri-1.0.1_Cont7.2, whole genome shotgun sequence. Available from http://www.ncbi.nlm.nih.gov/nuccore/NZ_ACBX02000037.1. AccessedApr. 11, 2012.
O’Sullivan DJ. 2000. Methods for analysis of the intestinal microflora. Curr. Issues Intest. Microbiol. 1: 39-50.
Petti C, Polage C, Schreckenberger P. 2005. The role of 16S rRNA gene sequencing in identification of microorganisms misidentified by conventional methods. J. Clin. Microbiol. 43:6123-6125.
Rinttilä T, Kassinen A, Malinen E, Krogius L, Palva A. 2004. Development of an extensive set of 16S rDNA-targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real-time PCR. J. Appl. Microbiol. 97: 11661177.
Songjinda P, Nakayama J, Kuroki Y, Tanaka S, Fukuda S, Kiyohara C, et al. 2005. Molecular monitoring of the developmental bacterial community in the gastrointestinal tract of Japan infants. Biosci. Biotechnol. Biochem. 69: 638-641.
Vanughan EE, Schut F, Heilig HG, Zoetendal EG, de Vos WM, Akkermans AD. 2000. A molecular view of the intestinal ecosystem. Curr. Issues Intest. Microbiol. 1: 1-12.
Wilson M. 2005. Microbial Inhabitant of Humans, pp. 2-3. 1st Ed. Cambridge University, New York.
Wu GD, Bushmanc FD, Lewis JD. 2013. Diet, the human gut microbiota, and IBD. Anaerobe 24: 117-120.
Wu GD, Chen J, Hoffmann C, Bittinger K, Chen Y, Keilbaugh SA, et al. 2011. Linking long-term dietary patterns with gut microbial enterotypes. Science 334: 105108.
Yatsuneko T, Rey FE, Manary MJ, Trehan I, DominguezBello MG, Contreras M, et al. 2012. Human gut microbiome viewed across age and geography. Nature 486: 222-227.
Zimmer J, Lange B, Frick JS, Sauer H, Zimmermann K, Schwiertz A, et al. 2012. A vegan or vegetarian diet substantially alters the human colonic faecal microbiota. Eur. J. Clin. Nutr. 66: 53-60.

Article Tools

Article

Research article

J. Microbiol. Biotechnol. 2014; 24(8): 1026-1033

Published online August 28, 2014 https://doi.org/10.4014/jmb.1310.10043

Senior Thai Fecal Microbiota Comparison Between Vegetarians and Non-Vegetarians Using PCR-DGGE and Real-Time PCR

Supatjaree Ruengsomwong ^{1, 2, 3}, Yuki Korenori ⁴, Naoshige Sakamoto ⁴, Bhusita Wannissorn ³, Jiro Nakayama ⁴ and Sunee Nitisinprasert ^{1, 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; Accepted: April 14, 2014

Abstract

The fecal microbiotas were investigated in 13 healthy Thai subjects using polymerase chain
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,
Clostridium colicanis, Eubacterium eligenes, E. rectale, Faecalibacterium prausnitzii, Megamonas
funiformis, Prevotella copri, and Roseburia intestinalis, belonging mainly to the groups of
Bacteroides, Prevotella, Clostridium, and F. prausnitzii. A dendrogram of the PCR-DGGE divided
the subjects; vegetarians and non-vegetarians. The fecal microbiotas were also analyzed using
a quantitative real-time PCR focused on Bacteroides, Bifidobacterium, Enterobacteriaceae,
Clostrium coccoides-Eubacterium rectale, C. leptum, Lactobacillus, and Prevotella. The nonvegetarian
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
Asians with similar diets.

Keywords: faecal microbiota, PCR-DGGE, quantitative real-time PCR, Bacteroides, Prevotella

Abstract

References

Arumugam M, Raes J, Pelletier E, Paslier DL, Yamada T, Mende DR, et al. 2011. Enterotypes of the human gut microbiome. Nature 473: 174-180.
Attasara P, Buasom R. 2012. Hospital-based Cancer Registry 2011, pp 1-4. Information Technology Division, National Cancer Institute, Bangkok.
Bartosch S, Fite A, Macfarlane GT, McMurdo ME. 2004. Characterization of bacterial communities in feces from healthy elderly volunteers and hospitalized elderly patients by using real-time PCR and effects of antibiotic treatment on the fecal microbiota. Appl. Environ. Microbiol. 70: 35753581.
Benno Y, Endo K, Mizutani T, Namba Y, Komori T, Mitsuoka T. 1989. Comparison of fecal microflora of elderly persons in rural and urban areas of Japan. Appl. Environ. Microbiol. 55: 1100-1105.
Claesson MJ, Jeffery IB, Conde S, Power SE, O’Connor EM, Cusack S, et al. 2012. Gut microbiota composition correlates with diet and health in the elderly. Nature 488: 178-184.
Clarridge J. 2004. Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clin. Microbiol. Rev. 17: 840-862.
Crowe FL, Roddam AW, Key TJ, Appleby PN, Overvad K, Jakobsen MU, et al. 2011. Fruit and vegetable intake and mortality from ischaemic heart disease: results from the European Prospective Investigation into Cancer and Nutrition (EPIC)-Heart study. Eur. Heart J. 465: 1-9.
De Filippo C, Cavalieri D, Di Paola M, Ramazzotti M, Poullet JB, Massart S, et al. 2010. Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. PNAS 107: 14691-14696.
Dethlefsen L, Eckburg PB, Bik EM, Relman DA. 2006. Assembly of the human intestinal microbiota. Trends Ecol. Evol. 21: 517-523.
Favier CF, Vaughan EE, de Vos WM, Akkermans AD. 2002. Molecular monitoring of succession of bacterial communities in human neonates. Appl. Environ. Microbiol. 68: 219-226.
Haarman M, Knol J. 2006. Quantitative real-time PCR analysis of fecal Lactobacillus species in infants receiving a prebiotic infant formula. Appl. Environ. Microbiol. 72: 23592365.
Hayashi H, Sakamoto M, Benno Y. 2002. Fecal microbial diversity in a strict vegetarian as determined by molecular analysis and culture cultivation. Microbiol. Immunol. 46: 819831.
Hayashi H, Sakamoto M, Kitahara M, Benno Y. 2003. Molecular analysis of fecal microbiota in elderly individuals 16S rDNA libraries and T-RFLP. Microbiol. Immunol. 47:5557-5570.
Hayashi H, Takahashi R, Nishi T, Sakamoto M, Benno Y. 2005. Molecular analysis of jejunal, ileal, caecal and recto sigmoidal human colonic microbiota using 16S rRNA gene libraries and terminal restriction fragment length polymorphism. J. Med. Microbiol. 54: 1093-1101.
Jeffery IB, O’Toole PW. 2013. Diet-microbiota interactions and their implications for healthy living. Nutrient 5: 234-252.
Kabeerdoss J, Devi RS, Mary RR, Ramakrishna BS. 2012. Faecal microbiota composition in vegetarians: comparison with omnivores in a cohort of young women in southern India. Br. J. Nutr. 108: 953-957.
Liszt K, Zwielehner J, Handschur M, Hippe B, Thaler R, Haslberger AG. 2009. Characterization of bacteria, Clostridia and Bacteroides in feces of vegetarians using qPCR and PCRDGGE fingerprinting. Ann. Nutr. Metab. 54: 253-257.
Mariat D, Firmesse O, Levenez F, Guimarães VD, Sokol H, Doré J, et al. 2009. The Firmicutes/Bacteroidetes ratio of the human microbiota changes with age. BMC Microbiol. 9: 123.
Matsuda K, Tsuji H, Asahara T, Matsumoto K, Takada T, Nomoto K. 2009. Establishment of an analytical system for the human fecal microbiota, based on reverse transcriptionquantitative PCR targing of multicopy rRNA molecules. Appl. Environ. Microbiol. 75: 1961-1969.
Matsuki T, Watanabe K, Fujimoto J, Takada T, Tanaka R. 2004. Use of 16S rRNA gene-targeted group-specific primers for real-time PCR analysis of predominant bacteria in human feces. Appl. Environ. Microbiol. 70: 7220-7228.
Maukonen J, Mättö J, Satokari R, Söderlund H, MattilaSandholm T, Saarela M. 2006. PCR DGGE and RT-PCR DGGE show diversity and short-term temporal stability in the Clostridium cocccoides-Eubacterium rectale group in the human intestinal microbiota. FEMS Microbiol. Ecol. 58: 517528.
Matijaši B B, O bermajer T, L ipoglavšek L, G rabnar I , Gorazd A, Rogelj I. 2013. Association of dietary type with fecal microbiota in vegetarians and omnivore in Slovenia. Eur. J. Nutr. DOI: 10.1007/s00394-013-0607-6.
Moore W, Moore L. 1995. Intestinal floras of populations that have a high risk of colon cancer. Appl. Environ. Microbiol. 61: 3202-3207.
Nair P, Mayberry JF. 1994. Vegetarianism, dietary fibre and gastro-intestinal disease. Digest. Dis. 12: 177-185.
Nam YD, Jung MJ, Roh SW, Kim MS, Bae JW. 2011. Comparative analysis of Korean human gut microbiota by pyrosequencing. PLoS ONE 6: e22109.
NCBI. Prevotella copri DSM 18205 P_copri-1.0.1_Cont7.2, whole genome shotgun sequence. Available from http://www.ncbi.nlm.nih.gov/nuccore/NZ_ACBX02000037.1. AccessedApr. 11, 2012.
O’Sullivan DJ. 2000. Methods for analysis of the intestinal microflora. Curr. Issues Intest. Microbiol. 1: 39-50.
Petti C, Polage C, Schreckenberger P. 2005. The role of 16S rRNA gene sequencing in identification of microorganisms misidentified by conventional methods. J. Clin. Microbiol. 43:6123-6125.
Rinttilä T, Kassinen A, Malinen E, Krogius L, Palva A. 2004. Development of an extensive set of 16S rDNA-targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real-time PCR. J. Appl. Microbiol. 97: 11661177.
Songjinda P, Nakayama J, Kuroki Y, Tanaka S, Fukuda S, Kiyohara C, et al. 2005. Molecular monitoring of the developmental bacterial community in the gastrointestinal tract of Japan infants. Biosci. Biotechnol. Biochem. 69: 638-641.
Vanughan EE, Schut F, Heilig HG, Zoetendal EG, de Vos WM, Akkermans AD. 2000. A molecular view of the intestinal ecosystem. Curr. Issues Intest. Microbiol. 1: 1-12.
Wilson M. 2005. Microbial Inhabitant of Humans, pp. 2-3. 1st Ed. Cambridge University, New York.
Wu GD, Bushmanc FD, Lewis JD. 2013. Diet, the human gut microbiota, and IBD. Anaerobe 24: 117-120.
Wu GD, Chen J, Hoffmann C, Bittinger K, Chen Y, Keilbaugh SA, et al. 2011. Linking long-term dietary patterns with gut microbial enterotypes. Science 334: 105108.
Yatsuneko T, Rey FE, Manary MJ, Trehan I, DominguezBello MG, Contreras M, et al. 2012. Human gut microbiome viewed across age and geography. Nature 486: 222-227.
Zimmer J, Lange B, Frick JS, Sauer H, Zimmermann K, Schwiertz A, et al. 2012. A vegan or vegetarian diet substantially alters the human colonic faecal microbiota. Eur. J. Clin. Nutr. 66: 53-60.

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Research article

Senior Thai Fecal Microbiota Comparison Between Vegetarians and Non-Vegetarians Using PCR-DGGE and Real-Time PCR

Abstract

Keywords

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Research article

Senior Thai Fecal Microbiota Comparison Between Vegetarians and Non-Vegetarians Using PCR-DGGE and Real-Time PCR

Abstract

References