Molecular Characterization ofBacteria l Communities inth eHuma n Gastrointestinal Tract Erwin G. Zoetendal Promoter: prof. dr.W . M.d eVo s Hoogleraarin de Microbiologic Co-promotor: dr.A .D .L .Akkerman s Universitairhoofddocent bij de leerstoelgroep Microbiologic Leden van de promotiecommissie: dr. T. Abee Wageningen Universiteit prof. dr.ir .M .W .A . Verstegen Wageningen Universiteit dr. G.W .Wellin g Rijksuniversiteit Groningen prof.dr .A .vo n Wright University ofKuopio, Finland -, "^ o o -• c Molecular Characterization ofBacteria l Communities inth e Human Gastrointestinal Tract Erwin G. Zoetendal Proefschrift ter verkrijging van degraa d van doctor opgeza gva n derecto r magnificus van Wageningen Universiteit, prof.dr . ir. L.Speelman , inhe topenbaa rt everdedige n opdinsda g 13novembe r 2001 desnamiddag st e 13.30uu r in deAula . SAJ l Yl \u~"? ISBN: 90-5808-520-1 Keywords:G Itract , bacterial community,bacteria l diversity, 16SrRNA ,TGGE ,DGGE , FISH. Thesis Wageningen University Research Centre,Wageningen , TheNetherland s - With References - With Summaryi nEnglis h and Dutch- 192 pages. •P. D ~/ pi .V?0\ ,5t Stellingen 1) Eeneiige tweelingen lijken ookwa t betreft hun bacterigle samenstelling in de darmmee r op elkaar dan op andere mensen. Dit proefschrift 2) Fermentatieve bacterign met relatief eenvoudige voedingsbehoeften zijn niet altijd gemakkelijk op een vaste voedingsbodem te kweken. Dit proefschrift 3) Door de enorme toename van het aantal 16S rDNA sequenties van ongecultiveerde bacterign in databanken, isdoo r het bos de boom nauwelijks nog te zien. Maidaketal. (2001) Nucleic Acids Res.29: 173-174 4) Op basis van enkelvoudige waamemingen kan geen uitspraak gedaan worden over PCR fouten. Wilsonand Blitchington (1996)Appl. Environ. Microbiol. 62: 2273-2278 Whitfordet al. (1998)Anaerobe 4: 153-163 5) Gezien de defmitie, is een reincultuur van bacterign niet te kweken. 6) Voor de medaille-ranglijst van de Olympische Spelen verdient het de aanbeveling om het aantal behaalde medailles door het aantal inwoners van hetbetreffend e land te delen. 7) Hoe dichter je bij Veenendaal komt, hoe vaker "Zoetendal" automatisch met twee a's wordt geschreven. Stellingen bij het proefschrift 'Molecular Characterization of Bacterial Communities in the Human Gastrointestinal Tract'. Erwin Zoetendal, Wageningen, 13Novembe r2001 . Contents Chapter 1 General introduction 1 Chapter 2 Molecular characterization of microbial communities based on 16S 11 rRNA sequence diversity Chapter 3 Temperature gradient gel electrophoresis analysis of 16S rRNA 47 from human fecal samples reveals stable and host-specific communities ofactiv ebacteri a Chapter 4 DNA isolation protocols affect the detection limit of PCR 65 approaches of bacteria in samples from the human gastrointestinal tract Chapter 5 Molecular diversity of Lactobacillus spp., and other lactic acid 77 bacteria in the human intestine as determined by specific amplification of 16Sribosoma l DNA Chapter 6 The host genotype affects the bacterial community in the human 101 gastrointestinal tract Chapter 7 The attached bacterial community from the human gastrointestinal 113 tract is uniformly distributed along the colon and differs from the fecal community Chapter 8 Quantification of uncultured Ruminococcus obeum-like bacteria in 129 human fecal samples with fluorescent insitu hybridization and flow cytometryusin g 16S ribosomal RNAtargete d probes Chapter 9 Victivallis vadensis gen. nov. sp. nov., a cellobiose-degrading 145 bacterium from human feces Chapter 10 Concluding remarks and future perspectives 157 Chapter 11 Summary 167 Chapter 12 Samenvatting 173 Publications 181 Nawoord 183 Curriculum vitae 185 Chapter 1 General Introduction The mammalian gastrointestinal (GI) tract can be regarded as an ecosystem, in which contributions of the microorganisms, epithelium, the immune system, and compounds that escaped digestion by the host have a major impact. This results in a very dynamic and complex ecosystem that is, however, difficult to study notably because of the complexity of the microbial community. This chapter will briefly summarize some characteristics of the GI tract and itsmicrobes ,whic h have lead toth e aimo f thisthesis . THE GI TRACT The GI tract can be regarded as a tube extending from the mouth to the anus which is divided into several well-defined anatomical regions. The GI tract has several functions from which the digestive and absorptive functions of nutritious compounds are most well known. After intake of food, the digestion starts in the mouth and compounds that can easily be digested by the host are already absorbed before they reach the large intestine. Enzymes, such as glycosidases, lipases, peptidases, and proteinases play a key role in the digestion of food compounds (reviewed by5) . The GI tract harbors abacteria l community consisting of approximately 1014 cells and therefore itoutnumber s the total cells of the human body by one order of magnitude (25). The epithelium of the GI tract is covered by a mucus layer which protects the GI tract against mechanical and enzymatical damage aswel l asbacteria l invasion. In addition, this continuous renewing mucus layer serves as an additional carbohydrate source for the bacterial community. The bacteria in the GI tract are not equally distributed. The stomach only harbors a small number of bacteria, because of the low pH (pH~2 when gastric acids are produced). The bacterial number in the small intestine is also low, because of the short transit time (~5h) of the contents. The highest bacterial number and diversity are found in the colon, because it has a relatively large transit time (-60 hours). It is estimated that this bacterial community makes upt o 55%o fth e fecal solids (reviewed by5) . The human colon is about 150 cm long with a surface area (undissected) of approximately 1300 cm2. It contains on average 220 g of contents from which the moisture content is approximately 86% in the caecum, falling to 77% in the sigmoid-rectum. The pH shifts from 5.4-5.9 in the beginning of the colon to 6.6-6.9 to the end. Therefore, the physiological conditions vary from beginning to the end of the colon. In the colon water and salts are absorbed. It is estimated that approximately 1.5 kg of material is entering the colon each day while approximately 120 g of stool are excreted each day. Because of bacterial fermentation of compounds that escaped digestion by the host the main end products found in thecolo n are acetate,propionate , andbutyrate . Theseshor tchai n fatty acids(SCFA )provid e a powerful driving force for movement of water out of the colonic lumen and consequently, may constitute an important protection against diarrhea. It is estimated that epithelial cells in the colon obtain 60-70%o fthei r energy from bacterial fermentation products (reviewed by5) . CULTIVATION OFG ITRAC T BACTERIA The GI tract harbors a diverse bacterial community. Since the description of "Bacterium coli commune"i n 1885 (7) many attempts have been made to isolate and characterize bacteria in the human GI tract and trying to understand their function. During the first half of the 20th century research of the bacteria in the GI tract was not so intense. An increase in interest on the GI tract bacteria appeared after several groups in the sixties started extensively studying their function in the GI tract. The major findings from this period and their importance have recently been summarized anddiscusse d (26). During the seventies the cultivation approaches were optimized after applying the anaerobic incubation techniques, developed by Hungate (17), to cultivate microorganisms from the human GI tract. A major increase in numbers of isolates from the GI tract has been achieved, notably by Moore, Holdeman, Finegold, and their colleagues (8,9 , 15, 16,23) . The most intensive culturing study performed has been described by Finegold and colleagues (9). In this 10 years during study fecal samples from 141 volunteers having various diets and disease states were analyzed. A high similarity in bacterial composition was found between the volunteers despite the differences in diet and disease state. These and other cultivation studies revealed many novel isolates, that the GI tract is dominated by strict anaerobic bacteria, that number of colony forming units per gram of wet weight feces reaches 1010 - 1011, and that the dominant community is relatively stable. Even up to now still novel species are being isolated from the human GI tract. It has been estimated that up to 400 bacterial species may be present in the GI tract of healthy volunteers (6). Although many variations in composition were found in various studies, the main genera which were commonly detected include Bacteroides, Bifidobacterium, Clostridium, Eubacterium, Fusobacterium, Peptostreptococcus, andRuminococcus (reviewed by 31). Since the different parts in the GI tract are hardly accessible, the majority of studies have beenperforme d using feces as inoculum. Only in some cases,th ebacteria l community at other parts in the GI tract have been studies by taking samples from biopsies and intestinal fluids, from sudden-death victims,o rdurin g surgery (2,3 , 11, 12, 18,21 , 37,reviewe d by9) . Although the culture-dependent studies gave many new insights in the distribution of the bacteria in the GI tract, they do not give a complete picture of the bacterial composition. For many ecosystems the so-called "great plate count anomaly" has been observed by comparing those counts and direct microscopic counts (4, 28). The culturability of bacteria from several habitats was found to vary between less than 1t o 15% (4). Similar comparisons have been made for bacteria from the human GI tract and it is estimated that between 10an d 50% can be obtained in culture (19, 22, 30, 35). Moore and colleagues reported that they could cultivate 93% of the total microscopic counts, although they mentioned that their microscopic counts is likely underestimated because of the staining procedure and counting clumps of cells instead of single cells (23).