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ORIGINAL ARTICLE Bacterial Growth and Metabolism on Surfaces in the Large Intestine S. Macfarlane, M. J. Hopkins and G. T. Macfarlane From the University of Dundee MRC Microbiology and Gut Biology Group, Ninewells Hospital and Medical School, Dundee, UK Correspondence to: G. Macfarlane University of Dundee, Department of Molecular and Cellular Pathology, Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland. Tel: » 44-1382 496250, Fax: » 44-1-1382 633952, E-mail: [email protected] Microbial Ecology in Health and Disease 2000; Suppl 2: 64– 72 The large intestinal microbiota is characteristically viewed as being a homogeneous entity, yet the proximal colon and distal bowel differ markedly in relation to their nutritional availabilities and physicochemical attributes. Moreover, individual species and assemblages of microorganisms exist in a multiplicity of different microhabitats and metabolic niches in the large gut, on the mucosa and in the mucus layer, as well as in the gut lumen. Examination of intestinal material by scanning electron microscopy and uorescent light microscopy shows that most of the bacteria are not freely dispersed, but occur in clumps, and in aggregates attached to plant cell structures and other solids. With respect to the numerically predominant species, bacteria attached to surfaces in the gut lumen appear to be phylogenetically similar but physiologically distinct from non-adherent populations. These adherent organisms are more directly involved in the breakdown of complex insoluble polymers than unattached bacteria, which provides a competitive advantage in the ecosystem. In healthy people, mucosal populations are more difcult to study than faecal bacteria due to difculty in gaining access to the bowel, and has restricted studies on these communities. Consequently, little information is available concerning the composition, metabolism and health-related signicance of bacteria growing at or near the mucosal surface. Key words: biolms, mucosa, bacterial metabolism, mucus, pathogens. SUBSTRATE AVAILABILITY AND ENERGY GENERATION IN THE MICROBIOTA Due to gastric acid and the washout effect resulting from rapid passage of digestive substances through the stomach and small bowel, the principal areas of permanent microbial colonisation of the human gut are the terminal ileum and large intestine. This is primarily a result of the slowing down of movement of digestive material in the colon, which provides time for a complex and stable microbial ecosystem to develop (1). The main dietary sources of carbon and energy for intestinal microorganisms are resistant starches, plant cell wall polysaccharides and oligosaccharides, together with peptides, proteins and other lower molecular weight substances that escape digestion and absorption in the small gut (2). These, together with proteins and other complex polymers formed by the host such as pancreatic secretions and mucopolysaccharides, are degraded by a wide range of hydrolytic enzymes to their component sugars and amino acids, which are then fermented by the bacteria. Through fermentation of carbohydrates (Fig. 1) and proteins (Fig. 2), and the absorption and utilisation of short chain fatty acids and other metabolic products, the large intestinal microora plays an important role in host digestive processes, enabling energy to be salvaged from unab© Taylor & Francis 2000. ISSN 1403-4174 sorbed dietary residues, as well as body tissues and secretions. Intestinal bacterial fermentations are regulated by the need to maintain redox balance, principally through the reduction and oxidation of ferredoxins, avins and pyridine nucleotides. To a large degree, this affects the ow of carbon through the bacteria, the energy yield obtained from the substrate, and the fermentation products that can be formed. Synthesis of reduced substances such as H2, lactate, succinate, butyrate and ethanol is used to effect redox balance during fermentation, whereas the production of more oxidized substances, such as acetate is associated with ATP generation. Conversely, more reduced fermentation products result in comparatively low ATP yields (3). Many of these electron sink products formed by carbohydrate fermenting bacteria, especially H2, succinate and lactate, serve as energy sources for non-saccharolytic species in the microbiota, thereby making a major contribution to species diversity in the ecosystem. Except in very broad terms, little is known of the metabolic relationships that exist between individual bacterial communities in the colon or of the ecology and multicellular organisation of the microbiota. A number of molecular studies have shown that only a small fraction of bacterial species in natural communities are culturable (4); Microbial Ecology in Health and Disease Bacterial growth on surfaces (5) thus, while we can determine that the ecosystem contains large numbers of phylogenetically and physiologically different bacteria, the relative population sizes and types of non-culturable organisms present in the microbiota are largely unknown. Many host and environmental factors affect the composition of the gut microbiota (see Table I), including dietary inuences, transit time of colonic contents and their pH (6). However, ecological and metabolic interactions between the bacteria themselves are also signicant, and are largely dependent on the type of environment in which the organisms exist. While bacterial species diversity largely derives from the multiplicity of carbon and energy sources available for fermentation in the colon, many different types of ecological interaction occur between intestinal microorganisms, including commensalism, where one species is stimulated by a second, which itself is unaffected by the growth and activities of the rst; neutralism, where bacterial communities co-exist but have no signicant metabolic effect on each other; antagonism, in which one population is repressed by inhibitory substances produced by another, and symbiosis, where two species have an obligate dependence on each other. Of particular importance is the ability to compete for limiting nutrients and, in some circumstances, adhesion sites on food particles, 65 colonic mucus or the mucosa. Unsuccessful contenders in these events are rapidly eliminated from the ecosystem. BIOFILMS IN THE LARGE GUT While the large intestinal microbiota is conveniently viewed as being a homogeneous entity, the reality is quite different, because the bacteria exist in a multiplicity of different microhabitats and metabolic niches that are associated with the mucosa, the mucus layer and particle surfaces in the colonic lumen. Furthermore, these microenvironments are constantly changing as resources are consumed or produced. Bacteria in the large bowel may occur individually, but it is more likely they exist in microcolonies or in associations with other species on the surfaces of particulate materials. In fact, where there are suitable surfaces, bacteria and other microorganisms seem to have an innate tendency to form biolms. In some circumstances, these structures may be composed of a single species, as in infections of catheters, heart valves and other medical prosthetics, but biolms usually comprise complex multi-species consortia that develop in response to the chemical composition of the substratum and other environmental determinants (7). Observational and modelling studies suggest that the initial colonisation of surfaces occurs through the attach- Fig. 1. Pathways of polysaccharide breakdown, and the major routes of fermentation of dietary carbohydrate in the gut microbiota, showing the principal end products of metabolism. NSP, non-starch polysaccharides; PK, phosphoketolase; TA, transaldolase. 66 S. Macfarlane et al. Fig. 2. Mechanisms of protein breakdown by bacteria in the large intestine and fates of the products of dissimilatory amino acid metabolism. ment of individual bacteria or small groups of organisms, followed by non-linear proliferation of the cells that ultimately leads to formation of the mature biolm. While there is still some debate on this subject, it appears that initial attachment occurs due to either electrostatic forces on the bacterial surface or to the production of a sticky glycocalyx by some cells. Bacteria growing in biolms often behave differently from their non-adherent counterparts, and in particular, the nature and efciency of their metabolism is changed, while many species exhibit greater resistance to antibiotics, and other inhibitory factors that have deleterious effects on planktonic bacteria (8– 10). Bacterial biolms occur in a variety of natural environments including sediments, soils, the oral cavity and the gastrointestinal tracts of animals, but their study has been neglected in the human gut. However, particle-associated and mucosal bacterial populations in the large bowel are probably components of highly evolved assemblages, analogous to those in oral biolm communities, where partner recognition appears to be very specic during the formative stages of co-aggregation (11). Biolm communities often exhibit highly coordinated multicellular behaviour, within and between species, and many biolm properties are dependent on local cell population densities. A good example of this is provided by quorum sensing transcriptional activation in Gram-nega- tive bacteria (12) (see also a review by Swift et al. in this supplement). Close spatial relationships between bacterial cells growing on surfaces are important in other ways, particularly in relation to metabolic communication between microorganisms in the microbiota. They are ecologically signicant in that they minimize potential growth limiting effects on secondary cross-feeding populations, that are associated with mass transfer resistance, as for example between H2-producing bacteria and H2-consuming syntrophs (13). This is apparent in Fig. 3 where an autouorescing microcolony of the archaeon Methanobrevibacter smithii can be seen growing in mucus. The obligate H2-utilising organism cannot digest mucus glycoprotein by itself and must therefore rely on H2 and CO2 formation during mucinolysis by saccharolytic and amino acid-fermenting bacteria growing in close proximity, to provide substrate. Another facet of bacterial growth in biolms in the colon is that species colonising surfaces in the gut lumen are likely to be more directly involved in the breakdown of complex insoluble polymeric substances than non-adherent organisms, giving them a signicant advantage in competing for nutrients in the ecosystem (14). MUCOSAL COMMUNITIES IN THE LARGE INTESTINE The compositions of mucosal bacterial communities in the gastrointestinal tracts of animals have been relatively well characterized, and in ruminants (15), termites (16) and chickens (17), specic microoras have been identied on epithelial surfaces. Secretory intestinal epithelia are covered in a mucus coating ranging between 100– 200 vm in Fig. 4. Scanning electron micrograph of bacterial microcolonies on the human rectal mucosa. The brous-like matrix is the mucus layer that has been dehydrated during sample preparation. Bacterial growth on surfaces Fig. 3. Light micrograph of an autouorescing microcolony of Methanobrevibacter smithii in intestinal mucus. Fig. 5. Bacteroides microcolonies on the rectal mucosa hybridized with a specic 16S rRNA probe labelled with Cy3. Fig. 6. Bidobacterium microcolonies hybridized with a specic 16S rRNA probe labelled with Cy5. 67 Fig. 7. Live:dead Baclight (SYTO 9:propidium iodide) stain of a rectal microcolony. Yellow cells are live, red cells are dead. Fig. 8. Spirochaete-like organisms on the colonic wall. The bacteria are stained with a eubacterial 16S rRNA probe, labelled with FITC. thickness (18), and the mucus gel seems to play a role in stabilising microbial communities growing in association with the mucosa (19). Due to the presence of facultative anaerobes, O2 does not appear to be a major factor affecting the growth of anaerobic organisms at the epithelial surface. Mucosal populations are difcult to study in healthy people, mainly because of the inaccessibility of the colon, and this has restricted work on these bacterial communities. Despite this, some reports suggest that gut epithelial populations in man are generally similar to those present in the gut lumen (20). Bacteroides occur on the mucosal surface, but other gut anaerobes including eubacteria, bidobacteria, clostridia and a variety of Gram-positive cocci are also present (21, 22). This can 68 S. Macfarlane et al. be seen in Fig. 4, which is an electron micrograph of the rectal epithelium, and in Figs. 5 and 6 which are light micrographs that respectively show Bacteroides and bidobacterial microcolonies on a rectal biopsy. Fig. 7 illustrates a live:dead stained rectal microcolony, comprising a mixture of live (yellow) and dead (red) cells. Other investigations involving the use of colonic and rectal biopsies have indicated that Bacteroides, particularly B. vulgatus and B. thetaiotaomicron, and bidobacteria, are the major anaerobes associated with mucosal surfaces in the gut (23– 25). Some mucosal bacteria that cannot be seen or cultured from lumenal contents have unusual morphological properties (26). Scanning electron micrographs of biopsy specimens show large helical cells, with lengths in excess of 60 vm residing in the mucus layer (21). A number of species growing in the mucus layer or in association with the epithelial surface are lamentous or spiral-shaped gliding bacteria (27). Fig. 8 is a uorescent light micrograph showing swarms of spirochaete-like organisms, unusually arranged in parallel on the surface of a colonic biopsy specimen. Mucin, other host secretions and epithelial cells may be particularly important substrates for these mucosal species. COLONISATION OF THE GUT MUCOSA The structure and composition of bacterial communities growing on the gut epithelium, as well as those existing in the mucus layer, are probably determined by a variety of host factors, including cellular and humoral immunity (IgA), together with elements of the innate immune system, e.g., antimicrobial peptides such as defensins, which are formed by polymorphonuclear cells and some enterocytes, and are active against Gram-positive and Gramnegative bacteria (28). The rate of synthesis and chemical composition of mucus, turnover rates of intestinal epithelial cells, availability of adhesion sites, lysozyme production, pancreatic secretions, especially pancreatic endopeptidases, colonisation resistance mediated by components of the microbiota and gut motility are also likely to be important. Bacteria growing on the epithelial surface also affect mucosal and systemic immunity in the host, involving intestinal epithelial cells, blood leukocytes, B and T lymphocytes, and other cells of the immune system (29). Bacterial products with immunomodulatory properties include lipoteichoic acids (LTA), endotoxic lipopolysaccharide and peptidoglycans (30). Bidobacterial LTA possess high binding afnity for human epithelial cell membranes, and also serve as carriers for other antigens, binding them to target tissues, where they provoke an immune reaction (31). Maintenance of immune system homeostasis depends to some degree on cell-cell contacts, intuitively therefore, bacteria colonising the epithelial surface would seem to be particularly involved in modulation of its activity. In addition, phenotypic and antigenic variation by the parasite are ongoing events during the colonisation process that often facilitates the evasion of host immune system surveillance (32, 33). It is also of interest that commensal and parasitic species living in close association with host tissues often directly exploit the nutritional potential of the substratum, examples include bacterial utilisation of complex host macromolecules such as mucins (34), as well as cell matrix constituents like vitronectin (35) and bronectin (36). Recent developments have demonstrated that some adhesive bacteria are able to recruit a variety of structurally diverse host proteins, adhesive glycoproteins, growth factors and cytokines, by initially binding heparin and functionally similar sulphated polysaccharides to their surfaces, whence they serve as non-specic, secondary recruiting sites for other host molecules (37). Several bidobacterial isolates of human origin have been reported to exhibit protein-mediated adherence to enterocyte-like Caco-2 cells in vitro (38). In these investigations, adherent B. longum, B. breve and B. infantis variously inhibited cell-association and invasion of several gut pathogens, including Escherichia coli, Salmonella typhimurium and Yersinia pseudotuberculosis. B. infantis and some strains of B. longum and B. breve seemed to attach strongly, while other B. breve and B. longum isolates were poorly adherent. With respect to other Gram-positive rods, some, though not all lactobacilli are able to attach to human intestinal epithelial cells (39). Species that colonize the gut in this way characteristically exhibit high surface hydrophobicities (40), although protein-mediated adherence also seems to play a role (41, 42). As with the bidobacteria, protein-dependent adherence of L. acidophilus to Caco-2 cells inhibited binding of S. typhimurium, Y. pseudotuberculosis and E. coli (43). Lactobacillus antimicrobial activity, stimulation of enterocyte production of antimicrobial substances with defensin-like characteristics, as well as occupation of enterocyte receptors by the lactobacilli were suggested as possible mechanisms associated with pathogen exclusion. Similar effects have been observed in vivo, where in human volunteer feeding trials, probiotic lactobacilli were observed to temporarily colonize the gut surface, and displace other organisms including clostridia and enterobacteria (44). Yogurt lactobacilli have also been observed to bind to circulating peripheral blood CD4 and CD8 T lymphocytes, though not to B cells (45), while lactobacilli that adhere to human gut epithelial cells (39) are capable of macrophage activation (46). PATHOGENIC BACTERIA AND MUCUS SURFACES IN THE GUT In the initial stages of colonisation or infection of mucosal surfaces in the large bowel, bacteria must be able to Bacterial growth on surfaces withstand the ow of the intestinal contents, and thereby avoid being physically removed from the epithelial surface (47). However, epithelial cells in the gut are covered by a layer of mucus, which prevents most microorganisms reaching the mucosal surface (48). This mucus forms a viscoelastic gel (49), and it is these gel-like properties that are mainly protective against adhesion and invasion by pathogenic microbes, bacterial toxins and end-products of metabolism, pancreatic endopeptidases, microbial antigens and other damaging agents present in the lumen of the bowel. Pathogenic bacteria in the gut deal with mucus barriers in different ways. For example, mucus has a protective role against Yersinia enterocolit ica by reducing binding of the organisms to brush border membranes (50), while sulphomucins prevent colonisation of the gastric mucosa by Helicobacter (51). In contrast, some Gram-negative pathogens that colonize the mouse intestine depend on their abilities to adhere to mucus (52). Several motile species are chemotactic (14, 53, 54) or possibly viscotaxic to mucin (55). Thus, virulent strains of Serpulina hyodysenteriae are considerably more chemotactic towards mucin than non-virulent isolates (56, 57). Moreover, some pathogens such as Campylobacter do not degrade mucus, but bind to the glycoprotein by means of specic adhesins, as a prelude to gaining access to cell membrane receptors (57). In other gut pathogens, mucus has an important nutritional function, and neuraminidase has been shown to be important for survival of Bacteroides fragilis in both in vivo and in vitro model systems (58). In some pathogens, colonisation of the mucosa is achieved following invasion of the overlying mucus, whilst others proceed through the mucus, adhering to and colonising underlying epithelial cells. The bacterial adhesins involved are diverse, but are usually cell surface protein structures, and the eukaryotic cell receptors are generally carbohydrate residues. It is likely that these pathogenic bacteria possess a number of different adhesins, that may be used at different stages in the infection process. For example, strains of E. coli can express several different mbriae which function as adhesins (see also review by Adlerberth et al. in this supplement). It has been suggested (59) that E. coli type 1 mbriae, which bind to D -mannose residues on eukaryotic cells, play an important role in colonisation of the urinary tract and large bowel. A high percentage of E. coli strains isolated from pyelonephritis produce a different type of structure termed P mbriae, while a variety of other bacteria also produce mbriae which are likely to function as adhesins, including Salmonella spp., Neisseria spp. and Pseudomonas spp (59). It has also been reported (60) that some strains of C. difcile are mbriated, but it remains to be determined whether these structures are associated with adhesion and pathogenesis. Using scanning electron microscopy Newell et al. (61), 69 observed in vitro adherence of C. jejuni to intestinal epithelial cells (62). Bacterial agella have been implicated as carrying adhesins of C. jejuni for eukaryotic cell (61); this was conrmed by studies of McSweegan and Walker (62) who also suggested the involvement of a number of bacterial adhesins, notably a bacterial surface protein. Other microbial adhesins that have been reported include the lamentous haemagglutinin of Bordetella pertussis (59), the mannose-resistant haemagglutinin of S. typhimurium (63), and the bacterial cell surface proteins that bind specically to bronectin on the surface of eukaryotic cells. Treponema pallidum, the aetiologic agent of syphilis, uses three surface adhesins (P1, P2, P3) to specically bind bronectin (59). Bacterial neuraminidases may also facilitate adherence by exposing receptors on the surface of eukaryotic cells. For example, the enzyme produced by Bacteroides fragilis has been suggested to expose a galactoside residue involved in adhesion (64). The capsule of B. fragilis is also believed to carry adhesins, as does the capsule in Shigella spp. where the adhesins are believed to be carbohydrate moieties (65). In addition to these specic interactions between bacterial adhesins and host cell receptors, non-specic mechanisms of cell attachment are also important. Hydrophobic interactions are thought to facilitate adhesion by overcoming repulsive forces between host and microbial cells, and are an important non-specic adhesion mechanism in mucosal association of bacteria. Indeed, many of the mbrial and non-mbrial adhesins described have a high surface hydrophobicity. It has been proposed that the adhesiveness of a pathogen is related to the hydrophobicity conferred by its surface structures, and that whatever the receptor attachment mechanism, surface hydrophobicity is involved (66). BACTERIA ADHERING TO DIGESTIVE RESIDUES IN THE GUT LUMEN AND MUCUS BREAKDOWN Salivary, bronchial, gastric, small and large intestinal mucins are all used as growth substrates by gut microorganisms. In small intestinal efuent, particulate substances such as partly digested plant cell materials are entrapped in a viscoelastic mucus gel, which must be broken down by bacteria in the colon to facilitate access to the food residues. Microscopic examination of lumenal contents or faecal material shows that the majority of bacteria are not freely dispersed, but occur in large clumps and in aggregates attached to plant cell structures and other solids. While few studies have been made on bacterial colonisation of particulate materials in the gut lumen in humans, microbiological analysis of partially digested food particles in faeces shows that biolm populations growing on the surfaces of particulate matter are members of complex multi-species consortia 70 S. Macfarlane et al. and, at genus level at least, the biolm populations are supercially similar to those in non-adherent microbiotas, with Bacteroides and bidobacteria predominating (67). Complete destruction of mucin and other complex polymers in the gut is completed through the activities of several different hydrolytic enzymes that can breakdown the protein backbone and carbohydrate side chains of the macromolecule. Many of these enzymes are catabolite regulated (67, 68) and their synthesis is therefore dependent on local concentrations of mucin and other inducer and repressor substances. While some intestinal bacteria form a wide range of glycoside hydrolases, which in theory enables them to completely digest heterogeneous polymers (69, 70), evidence suggests that the breakdown of mucin and other complex organic molecules in the bowel is a cooperative activity. In contrast to the depolymerisation of mucin and other host mucopolysaccharides, there is undoubtedly severe competition between gut bacteria for the products of oligosaccharide hydrolysis. In fact, there are substantial populations of saccharolytic bacteria on the rectal mucosa that are unable to digest the glycoprotein by themselves and they must exist by cross-feeding on carbohydrate fragments produced by mucinolytic species growing in nearby microcolonies (71). Enzyme measurements have indicated that biolms occurring on digestive substances in the gut lumen form metabolically distinct assemblages in the ecosystem with respect to the breakdown and metabolism of mucins and other complex macromolecules (67). It was shown in these studies that, with the exception of N-acetyl hgalactosaminidase, the vast majority of mucinolytic glycosidases were cell-associated in faecal material. Whilst there was little difference in b -galactosidase and N-acetyl b-glucosaminidase in the biolms, h-fucosidase and N-acetyl h-galactosaminidase activities were manifestly lower than in non-adherent populations. Proteases and peptidases must also take part in mucin digestion, and measurements of peptidolytic enzymes in lumenal biolm and non-adherent populations, using a range of protease inhibitors, indicated that while the spectrum of proteolytic:peptidolytic activity was broadly similar in both microbiotas (e.g. with respect to serine, thiol and aspartic protease proles), there were variations in trypsin, chymotrypsin and to a lesser extent, metalloprotease activities. The higher trypsin and chymotrypsin in the biolms were attributed to adsorbed pancreatic endopeptidases, whereas lower metalloprotease activities were thought to reect differences in bacterial enzyme expression. Valyl alanyl:glycyl prolyl arylamidase, which can be formed in very high levels by members of the Bacteroides fragilis group (34), was also markedly lower in lumenal biolms. To conclude, because few studies have been made, little information is available concerning the structure and function of bacterial biolms in the large intestine or their metabolic signicance to the host. Moreover, because the investigation of bacterial growth on surfaces and in aggregates is still in its infancy, many of the analytical tools that have been used to study biolm composition and metabolism are innately destructive and do not provide much information on organisation and community structure in biolms. However, the developing shift in emphasis away from culture-based studies, and the introduction of methodologies involving measurements of ribosome abundance, using quantitative hybridisation techniques (72) should facilitate future work on gut biolms. Quantitation of both total rRNA and that relating to specic populations together with uorescent labelling (73) and whole cell hybridisation: confocal microscopy (74) will improve our understanding of the temporal, metabolic and spatial organisation of these microcosms. ACKNOWLEDGEMENTS This review has been carried out with nancial support from the Commission of the European Communities, Agriculture, and Fisheries (FAIR), specic RTD programme PL98-4230 ‘Intestinal Flora: Colonization Resistance and Other effects’. It does not reects its views and in no way anticipates the Commission’s future policy in this area. The careful assistance of Donatella Lombardi in preparation of the manuscript is gratefully acknowledged. REFERENCES 1. Cummings JH. Diet and transit through the gut. J Plant Foods 1978; 3: 83– 95. 2. Cummings JH, Macfarlane GT. The control and consequences of bacterial fermentation in the human colon-a review. J Appl Bacteriol 1991; 70: 443– 59. 3. Macfarlane GT, Gibson GR. (1996). Carbohydrate fermentation, energy transduction and gas metabolism in the human large intestine. In: Mackie RI, White BA (ed) Ecology and Physiology of Gastrointestinal Microbes vol. 1: Gastrointestinal Fermentations and Ecosystems. Chapman & Hall, New York, pp. 269– 318. 4. Dunbar J, White S, Forney L. Genetic diversity through the looking glass: effect of enrichment bias. Appl Environ Microbiol 1997; 63: 1326– 31. 5. Hugenholtz P, Pace NR. Identifying microbial diversity in the natural environment: a molecular phylogenetic approach. Trends Biotechnol 1996; 14: 190– 7. 6. Macfarlane GT, Cummings JH. (1991). The colonic ora, fermentation and large bowel digestive function. In: Phillips SF, Pemberton JH, Shorter RG (ed) The Large Intestine: Physiology, Pathophysiology and Disease. Raven Press, New York, pp. 51– 92. 7. Bradshaw DJ, Marsh PD, Watson K, Allison C. (1997). Inter-species interactions in microbial communities. In: Wimpenny J, Handley P, Gilbert P, Lappin-Scott H, Jones M (ed) Biolms. Community Interactions and Controls. Bioline, Cardiff, pp. 63– 71. Bacterial growth on surfaces 8. Anwar H, Dasgupta MK, Costerton JW. Testing the susceptibility of bacteria in biolms to antibacterial agents. Antimicrob Agents Chemotherapy 1990; 34: 2043– 6. 9. Mozes N, Rouxhet PG. (1992) Inuence of surfaces on microbial activity. In: Melo LF, Bott TR, Capdeville B (ed) Biolms-Science and Technology. Kluwer Academic Publishers, Doorderecht, pp. 125–136. 10. Van Loosdrecht MCM, Lyklema J, Norde W, Zehnder AJB. Inuence of interfaces on microbial activity. Microbiol Rev 1990; 54: 75– 87. 11. Kolenbrander PE. Surface recognition among oral bacteria: multigeneric coaggregations and their mediators. Crit Rev Microbiol 1989; 17: 137– 59. 12. Salmond GP, Bycroft BW, Stewart GS, Williams P. The bacterial ‘enigma’: cracking the code of cell-cell communication. Mol Microbiol 1995; 16: 615–24. 13. Conrad R, Phelps TJ, Zeikus JG. Gas metabolism evidence in support of the juxtaposition of hydrogen-producing and methanogenic bacteria in sewage sludge and lake sediments. Appl Environ Microbiol 1985; 50: 595– 601. 14. Macfarlane GT, Macfarlane S, Sharp R. Differential expression of virulence determinants in Clostridium septicum in relation to growth on mucin and the swarm cell cycle. Biosci Microbiol 1997; 16: 28. 15. Wallace RJ, Cheng K-J, Dinsdale D, Orskov ER. An independent microbial ora of the epithelium and its role in the microbiology of the rumen. Nature 1979; 279: 424– 6. 16. Breznak JA, Pankatz HS. In situ morphology of the gut microbiota of wood eating termites [Reticulitennes aviceps Kollar and Coptotermes formosanus Shiraki]. Appl Environ Microbiol 1977; 33: 406– 26. 17. Lee A. (1980). Normal ora of animal intestinal surfaces. In: Bitton G, Marshall KC (ed) Adsorption of Microorganisms to Surfaces. John Wiley, New York, pp. 145– 174. 18. Pullen RD, Thomas GAO, Rhodes M, Newcombe RG, Williams GT, Allen A, Rhodes J. Thickness of adherent mucus gel on colonic mucosa in humans and its relevance to colitis. Gut 1994; 35: 353– 9. 19. Savage DC. Factors involved in colonization of the gut epithelial surface. Am J Clin Nutr 1978; 31: S131– 5. 20. Nelson DP, Mata LJ. Bacterial ora associated with the human gastrointestinal mucosa. Gastroenterology 1970; 58: 56– 61. 21. Croucher SC, Houston AP, Bayliss CE, Turner RJ. Bacterial populations associated with different regions of the human colon wall. Appl Environ Microbiol 1983; 45: 1025– 33. 22. Edmiston CE jr, Avant GR, Wilson FA. Anaerobic bacterial populations on normal and diseased human biopsy tissue obtained at colonoscopy. Appl Environ Microbiol 1982; 43: 1173– 81. 23. Macfarlane S, Cummings JH, Macfarlane GT. (1999) Chemotaxonomic analysis of bacterial populations on the human rectal mucosa. Proceedings of the XXIV International Congress on Microbial Ecology and Disease. San Francisco, p. 5.016. 24. Macfarlane S, Cummings JH, Macfarlane GT. (2000). Bacterial populations on the rectal mucosa in healthy and colitic subjects. Proceedings of the American Gastroenterological Association. San Diego, A-131. 25. Poxton IR, Brown R, Sawyerr AF, Ferguson A. The mucosal anaerobic Gram-negative bacteria of the human colon. Clin Inf Dis 1997; 25: S111– 3. 26. Lee FD, Kraszewski A, Gordon J, Howie JGR, McSeveney D, Harland WA. Intestinal spirochaetosis. Gut 1971; 12: 126– 33. 71 27. Takeuchi A, Jervis HR, Nakagawa H, Robinson DM. Spiralshaped organisms on the surface colonic epithelium of the monkey and man. Am J Clin Nutr 1974; 27: 1287– 96. 28. Mahida YR, Rose F, Chan WC. Antimicrobial peptides in the gastrointestinal tract. Gut 1997; 40: 161– 3. 29. Schiffrin EJ, Brassart D, Servin AL, Rochat F, DonnetHughes A. Immune modulation of blood leukocytes in humans by lactic acid bacteria: criteria for strain selection. Am J Clin Nutr 1997; 66: 15S– 20S. 30. Standiford TK, Arenberg DA, Danforth JM. Lipoteichoic acid induces secretion of interleukin-8 from human blood monocytes: a cellular and molecular analysis. Infect Immun 1994; 62: 119–25. 31. Op den Camp HJM, Oosterhof A, Veerkamp JH. Interaction of bidobacterial lipoteichoic acid with human intestinal epithelial cells. Infect Immun 1985; 47: 332– 4. 32. Finlay BB, Falkow S. Common themes in microbial pathogenicity revisited. Microbiol Mol Biol Rev 1997; 61: 136– 69. 33. Rostrand KS, Esko JD. Microbial adherence to and invasion through proteoglycans. Infect Immun 1997; 65: 1 – 8. 34. Macfarlane GT, Gibson GR. Formation of glycoprotein degrading enzymes by Bacteroides fragilis. FEMS Microbiol Lett 1991; 77: 289– 94. 35. Dehio M, Gomez-Duarte OG, Dehio C. Vitronectin-dependent invasion of epithelial cells by Neisseria gonorrhoea involves alpha(v) integrin receptors. FEBS Lett 1998; 424: 84– 8. 36. Patti JM, Allen BL, McGavin MJ. MSCRAMM-mediated adherence of microorganisms to host tissues. Annu Rev Microbiol 1994; 48: 585– 617. 37. Duensing TD, Wing JS, van Putten JPM. Sulfated polysaccharide-directed recruitment of mammalian host proteins: a novel strategy in microbial pathogenisis. Infect Immun 1999; 67: 4463– 8. 38. Bernet MF, Brassart D, Neeser J-R, Servin AL. Adhesion of human bidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions. Appl Environ Microbiol 1993; 59: 4121– 8. 39. Kleeman EG, Klaenhammet TR. Adherence of Lactobacillus species to human fetal intestinal cells. J Dairy Sci 1982; 65: 2063– 9. 40. Wadstrom T, Andersson K, Sydow M, et al. Surface properties of lactobacilli isolated from the small intestine of pigs. J Appl Bacteriol 1987; 62: 513– 20. 41. Chauviere G, Coconnier M-H, Kerneis S, Fourniet J, Servin AL. Adhesion of human Lactobacillus acidophilus strain LB to human enterocyte-like Caco-2 cells. J Gen Microbiol 1992; 138: 1689– 96. 42. Reid G, Servin A, Bruce AW, Busscher HJ. Adhesion of three Lactobacillus strains to human urinary and intestinal epithelial cells. Microbios 1993; 75: 57– 65. 43. Bernet MF, Brassart J, Neeser JR, Servin AL. Lactobacillus acidophilus LA1 binds to cultured human intestinal cell lines and inhibits cell attachment and cell invasion by enterovirulent bacteria. Gut 1994; 35: 483– 9. 44. Johansson M-L, Molin G, Jeppsson B, Nobaek S, Ahrne S, Bengmark S. Administration of different Lactobacillus strains in fermented oatmeal soup: in vivo colonization of human intestinal mucosa and effect on the indigenous ora. Appl Environ Microbiol 1993; 59: 15–20. 45. De Simone C, Grassi PP, Bianchi-Salvadori B, Miragliotti G, Vesely R, Jirillo E. Adherence of specic yogurt microorganisms to human peripheral blood lymphocytes. Microbios 1988; 55: 49– 57. 72 S. Macfarlane et al. 46. Perdigon G, Nader De Macios ME, Alvarez S, Oliver G, Pesce AA, Ruiz Holgado AAP. Effect of perorally administered lactobacilli on macrophage activation in mice. Infect Immun 1986; 53: 404– 10. 47. Smith H. The revival of interest in mechanisms of bacterial pathogenicity. Biol Rev 1995; 70: 277– 316. 48. Florey H. Mucin and the protection of the body. Proc Royal Soc (London) 1955; 143: 144– 58. 49. Allen A. (1981). Structure and function of gastrointestinal mucus. In: Johnson LR (ed) Physiology of the Gastrointestinal Tract. Raven Press, New York, pp. 617– 639. 50. Mantle M, Basaraba L, Peacock SC, Gall DG. Binding of Yersinia enterocolitica to rabbit brush border membranes, mucus, and mucin. Infect Immun 1989; 57: 3292–9. 51. Piotrowski J, Slomiany A, Murty VLN, Kekete Z, Slomiany BL. Inhibition of Helicobacter pylori colonization by sulfated gastric mucin. Biochem Int 1991; 24: 749– 56. 52. Cohen PS, Wadolkowski EA, Laux DC. Adhesion of a human fecal Escherichia coli strain to a 50.5 KDal glycoprotein receptor present in mouse colonic mucus. Microecol Ther 1986; 16: 231– 41. 53. Freter R, O’Brien PCM. The role of chemotaxis in the association of motile bacteria with intestinal mucosa: in vitro studies. Infect Immun 1981; 34: 234– 40. 54. Lee SG, Changsung K, Young CH. Successful cultivation of a potentially pathogenic coccoid organism with trophism for gastric mucin. Infect Immun 1997; 65: 49– 54. 55. Wilson LM. (1997). Physiological Studies on Swarming and Production of Virulence Determinants in Clostridium septicum. Ph.D Thesis, Cambridge University, Cambridge. 56. Milner JA, Sellwood R. Chemotactic response to mucin by Serpulina hyodysenteriae and other porcine spirochetes: Potential role in intestinal colonization. Infect Immun 1994; 62: 4095– 9. 57. Sylvester FA, Philpott D, Gold B, Lastovica A, Forstner JF. Adherence to lipid and intestinal mucin by a recently recognized human pathogen, Campylobacter upsaliensis. Infect Immun 1996; 64: 4060– 6. 58. Godoy VG, Dallas MM, Russo TA, Malamy MH. A role for Bacteroides fragilis neuraminidase in bacterial growth in two model systems. Infect Immun 1993; 61: 4415– 26. 59. Finlay BB, Falkow S. Common themes in microbial pathogenicity. Microbiol Rev 1989; 53: 210– 30. 60. Borriello SP, Davies HA, Barclay FE. Detection of mbriae amongst strains of Clostridium difcile. FEMS Microbiol Lett 1988; 49: 65– 7. 61. Newell DB, McBride H, Dolby JM. (1983). The signicance of agella in the pathogenesis of Campylobacter jejunii. In: Pearson AD, Skirrow MB, Rowe B (ed) Campylobacter II. Public Health Laboratory Service, London, p. 109. 62. McSweegan E, Walker RI. Identication and characterisation of two Campylobacter jejuni adhesins for cellular and mucous substrates. Infect Immun 1986; 53: 141– 8. 63. Halula MC, Stocker BAD. Distribution and properties of the mannose-resistant hemagglutinin produced by Salmonella species. Microb Pathol 1987; 3: 455–9. 64. Guzman CA, Plate M, Pruzzo C. Role of neuraminidase-dependent adherence in Bacteroides fragilis attachment to human epithelial cells. FEMS Microbiol Lett 1990; 71: 187– 92. 65. Haque MA, Qadri F, Ohki K. Surface components of Shigella involved in adhesion and haemagglutination. J Appl Bacteriol 1995; 79: 191–6. 66. Burke DA, Axon ATR. Hydrophobic adhesion of E. coli in ulcerative colitis. Gut 1988; 29: 41– 3. 67. Macfarlane S, McBain AJ, Macfarlane GT. Consequences of biolm and sessile growth in the large intestine. Adv Dent Res 1997; 11: 59– 68. 68. Macfarlane GT, Hay S, Gibson GR. Inuence of mucin on glycosidase, protease and arylamidase activities of human gut bacteria grown in a 3-stage continuous culture system. J Appl Bacteriol 1989; 66: 407– 17. 69. Degnan BA. (1993). Transport and Metabolism of Carbohydrate by Anaerobic Gut Bacteria. Ph.D Thesis, Cambridge University, Cambridge. 70. Pettipher GL, Latham M. Production of enzymes degrading plant cell walls and fermentation of cellobiose by Ruminococcus avifaciens. J Gen Microbiol 1979; 110: 29– 38. 71. Macfarlane GT, Cummings JH, Macfarlane S. (1999). Bacterial biolms in the large intestine. Proceedings of the 179th Meeting of the Pathological Society of Great Britain and Ireland. Dundee, A131. 72. Stahl DA, Amman RI. (1991). Development and application of nucleic acid probes in bacterial systematics. In: Stackenbrandt E, Goodfellow M (ed) Sequencing and Hybridization Techniques in Bacterial Systematics. John Wiley, Chichester, pp. 205– 248. 73. Amman RI, Krumholz L, Stahl DA. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic and environmental studies in microbiology. J Bacteriol 1990; 172: 762– 70. 74. Wolfaardt GM, Lawrence JR, Robarts RD, Caldwell SJ, Caldwell DE. Multicellular organisation in a degradative biolm community. Appl Environ Microbiol 1994; 60: 434– 46.