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In vitro inhibition of adhesion of Escherichia coli Arthur Ouwehand Department of General and Marine Microbiology Göteborg University Göteborg Sweden 1 In vitro inhibition of adhesion of Escherichia coli In vitro inhibition of adhesion of Escherichia coli. Arthur C. Ouwehand 2 Department of General and Marine Microbiology Göteborg University Göteborg Sweden 3 Arthur C. Ouwehand 4 In vitro inhibition of adhesion of Escherichia coli 1996 ISBN 91-628-2194-6 5 In vitro inhibition of adhesion of Escherichia coli. Arthur C. Ouwehand Akademisk avhandling. för filosofie doktorsexamen i mikrobiologi (examinator Professor Lennart Adler), som enligt biologiska sektionsstyrelsens beslut kommer att offentlig försvaras den 1 november 1996, kl. 10.00 i föreläsningssal A1024 Ivan Ivarsson, Medicinaregatan 3B, Göteborg. Göteborg 1996 ISBN 91-628-2194-6 6 Voor mam en pap 7 In vitro inhibition of adhesion of Escherichia coli Arthur C. Ouwehand Department of General and Marine Microbiology, Göteborg University S-413 90 Göteborg, Sweden. Abstract. For many Escherichia coli strains, adhesion to tissues, is considered a prerequisite for pathogenesis. Inhibition of this adhesion, may therefore prevent the establishment of disease in an early stage. The aim of this work was to identify substances that may inhibit the in vitro binding to intestinal mucus of pathogenic E. coli strains expressing either K88 or SfaII fimbriae. Lactobacillus fermentum strain 104r had been shown previously to produce a substance inhibitory to K88 mediated adhesion. The active component and the mechanism of inhibition have been investigated. Because human milk contains substances that inhibit pathogen adhesion and antimicrobial substances have been detected in bovine colostrum, the capacity of bovine colostrum constituents to inhibit SfaII-fimbriaemediated adhesion was investigated. L. fermentum 104r spent culture liquid and bovine colostrum were subjected to size exclusion chromatography and ultrafiltration followed by anion exchange chromatography. The fractions showing adhesion inhibitory activity were treated with different enzymes and chemicals in order to identify the active components and determine their modes of action. A range of Lactobacillus strains were tested for their ability to produce similar adhesion inhibitory activity. Active components, from Lactobacillus spent culture liquid and bovine colostrum, were examined for activity against other enteropathogens. It was found that L. fermentum 104r spent culture liquid contained a 1700 K carbohydrate that mediated the observed inhibition of K88 fimbrial adhesion. The carbohydrate was concluded to be a cell wall fragment, since the activity was affected by lysozyme treatment. No direct evidence could be found for the binding of the carbohydrate to the intestinal mucus. Indirect evidence suggests blocking of K88 receptors by steric hindrance, however, the precise mode of action still remains to be elucidated. It was observed that other Lactobacillus strains of intestinal origin also produced inhibitory components and that the greatest inhibition was noted for different K88 variants and SfaII fimbriae. Interestingly, the L. fermentum 104r spent culture liquid contained a high molecular weight substance capable of enhancing the antimicrobial activity of organic acids against E. coli growth in vitro. Its identity remains however to be determined. It was demonstrated that bovine colostrum contained a substance that inhibited SfaII fimbriae mediated adhesion with some effect on SfaI fimbrial adhesion. The substance was identified as -lactoglobulin, a major milk whey protein. -lactoglobulin binds in a concentration dependent 8 manner to intestinal mucus; using multiple receptors, and blocks, as proposed for Lactobacillus spent culture liquid, adhesion sites by steric hindrance. The activity of -lactoglobulin was found to be heat stable and dependant upon the presence of 3 disulfide bridges. In all cases, the in vivo importance of these findings remains to be determined. It was concluded that bovine colostrum and Lactobacillus spent culture fluid contain components inhibitory to adhesion on K88 and SfaII fimbriae. The role of this inhibition in pathogenicity is addressed in this thesis. Key words: Lactobacillus; Cell wall; Bovine colostrum; -lactoglobulin; Fimbriae; ETEC Göteborg 1996 ISBN 91-628-2194-6 9 In vitro inhibition of adhesion of Escherichia coli by Arthur C. Ouwehand Department of General and Marine Microbiology, Göteborg University Medicinaregatan 9C, S-413 90 Göteborg, Sweden This thesis is based on the following papers, which are referred to in the text by their respective Roman numerals: I Rojas, M., A.C. Ouwehand and P.L. Conway. Interactions between high and low molecular weight compound(s) in Lactobacillus spent culture fluid with antagonistic against E. coli K88 growth. Submitted II Ouwehand, A.C. and P.L. Conway. 1996. Purification and characterization of a component produced by Lactobacillus fermentum that inhibits the adhesion of K88 expressing Escherichia coli to porcine ileal mucus. J. Appl. Bact. 80:311-318. III Ouwehand, A.C. and P.L. Conway. 1996. Specificity of spent culture fluids of Lactobacillus sp. to inhibit adhesion of enteropathogenic Escherichia coli cells. Microb. Ecol. Health Dis. in press IV Ouwehand, A.C., P.L. Conway and S.J. Salminen. 1995. Inhibition of S-fimbria-mediated adhesion to human ileostomy glycoproteins by a protein isolated from bovine colostrum. Infect. Immun. 63:49174920. V Ouwehand, A.C., S.J. Salminen, M. Skurnik and P.L. Conway. 1996. Inhibition of enteropathogen adhesion by -lactoglobulin. Submitted to Infect. Immun. 10 Contents Page 1 Introduction 1.1 General 1.2 Aims 1 1 1 2 The digestive system 2.1 Anatomy and morphology 2.1.1 The mouth 2.1.2 The oesophagus 2.1.3 The stomach 2.1.4 The small intestine 2.1.5 The large intestine, colon and caecum 2.2 Mucus 2.2.1 Function 2.2.2 Composition 2.3 The normal microflora 2.3.1 The mouth 2.3.2 The oesophagus 2.3.3 The stomach 2.3.4 The small intestine 2.3.5 The large intestine 2.4 Enteric pathogens 2.4.1 General 2.4.2 E. coli 3 3 3 3 3 5 6 6 6 7 8 8 9 9 10 10 11 11 11 3 Milk 3.1 Composition and functions 3.2 Milk whey 3.2.1 Oligosaccharides 3.2.2 Proteins 13 13 13 13 14 4 Lactobacilli17 4.1 General background 4.2 Proposed functions in the intestine 4.2.1 Colonisation resistance 17 17 18 5 Adhesion 5.1 General background 5.2 Non-specific adhesion 5.3 Specific adhesion 21 21 21 23 11 5.4 Adhesion assays 25 6 Inhibition of adhesion 6.1 General background 6.2 Affecting the adhesin 6.2.1 Blocking the adhesin 6.2.2 Reducing adhesin expression 6.3 Affecting the receptor 6.3.1 No receptor 6.3.2 Modifying the receptor 6.3.3 Specific blocking of the receptor 6.3.4 Colonisation resistance. Non-specific blocking of the receptor 6.3.5 Lactobacillus cell wall fragments. Non-specific blocking of the receptor 6.3.6 -lactoglobulin. Non-specific blocking of the receptor 27 27 27 27 29 30 30 30 31 7 Conclusions 40 8 Acknowledgements 42 9 References 43 31 33 37 1 Introduction 1.1 General Many pathogens basically cause only one type of disease. Escherichia coli strains, however, are able to cause a variety of different diseases; diarrhoea, urinary tract infection, sepsis, meningitis etc. In order to cause disease, pathogens possess so-called virulence factors. These include special structures that facilitate attachment to host tissues, enzymes and other proteins that allow invasion and translocaton, production of toxins, capsules etc. 159. One of the first steps in the development of disease is adhesion of the pathogen to host tissue 9, 59, 93, 152. This is especially important in an environment as the small intestine. Once a pathogen has adhered, it can start producing toxins; which can induce diarrhoea, or it can invade and even translocate and cause sepsis and infect other organs. The newborn, receives many protective factors from the milk, among them antibodies. These passively protect the newborn. Upon weaning, this protection seizes. When this weaning is abrupt and early, and accompanied with other forms of stress, as in commercial pig raising, it leaves the young 12 animal very susceptible to pathogens. One of the pathogens commonly causing disease in piglets is K88 fimbriae bearing E. coli 98. Large scale use of antibiotics is undesirable and vaccination against post-weaning diarrhoea is still not effective. Other ways of controling enterotoxigenic E. coli are therefore of interest. Inhibition of adhesion may be an alternative strategy. Extraintestinal E. coli strains are able to cause severe sepsis and newborn meningitis 82. Over 30% of all cases of sepsis have been found to be caused by E. coli 64. E. coli expressing SfaII fimbriae are a major cause of newborn meningitis and sepsis 81. Of the investigated strains isolated from newborn meningitis, 30% produced SfaII fimbriae 104. Here too, inhibiton of adhesion might provide a way of preventing infection at an early stage. 1.2 Aims The aim of this thesis was to investigate factors that might interfere with the initial binding of K88 fimbriae and SfaII fimbriae expressing E. coli. Component(s) produced by Lactobacillus fermentum strain 104r had previously been found to inhibit K88 mediated adhesion 14. The aim was to purify and characterise the component(s), investigate the mode of action and characterise its spectrum of activity in vitro. The gastrointestinal tract and the oropharynx have been shown to be the main reservoir for E. coli that are able to translocate 80. Human milk has been found to contain substances that can inhibit the adhesion of SfaII fimbriated E. coli. Since bovine colostrum is a rich source of growth factors, antimicrobials etc. It might also contain similar adhesion inhibitory substances. These substances might inhibit the colonisation of these organs, thereby reducing the risk of translocation, from the digestive tract to the blood, and disease. 13 2 The digestive system 2.1 Anatomy, morphology and physiology. The main function of the digestive system is the digestion of food (mouth and especially in the stomach and the small intestine) 188, absorption of nutrients and water (small and large intestine) 192 and the storage of wastes (large intestine) 188. The anatomy, morphology and physiology of the gastrointestinal tract exert a large influence on the normal microflora and possible enteropathogens that live in the intestine. In addition, colonisation of the intestine by the pathogens will be influenced by these factors. 2.1.1 The Mouth In the mouth, food is mechanically broken down into small fragments by mastication and mixed with saliva. Saliva contains mainly water. The major electrolytes are Na+, K+, Cl- and HCO3-, the latter functions as a buffer. Mucus is one of the main organic constituents of saliva, it lubricates and protects the mucosal surfaces. Digestive enzymes in saliva are -amylase which hydrolyses -1,4 glucosidic linkages, and lipase which especially in the neonate is important for the hydrolysis of triglycerides. Other enzymes found in the mouth include lactoperoxidase which scavenges H2O2 (its activity will be discussed in section 3.2.2e), and lysozyme 163 which can act as an antimicrobial substance by hydrolysing the -1,4 bond between N-acetylglucosamine and N-acetylmuramate in the bacterial cell wall 184. In addition to smooth and rough squamous epithelium, the mouth also contains hydroxyapatite on the tooth surfaces to which bacteria can adhere 188. 2.1.2 The oesophagus The oesophagus is a hollow tube that transports the food from the mouth to the stomach with waves of contraction passing along its length. The peristaltic wave lasts for 7-10 seconds, but due to gravity, food may descend more rapidly 163. The oesophagus is lined with keratinized squamous epithelium, and may contain sparsely distributed mucus producing cells 78. 14 2.1.3 The stomach The stomach is a curved organ which receives ingested food and can store it temporarily. In humans it can accommodate up to 5 liters. It functions as a mixing and digestive organ. In the stomach, the ingested food is transformed into a fairly homogenous product, in terms of pH, osmolality, consistency and temperature. The central, and major, part of the stomach is called the body or corpus. The region where the oesophagus enters the stomach is called the cardia. In pigs, this area is called the pars oesophagus 98. Next to the cardia/pars oesophagus, is the upper most region of the stomach, namely the fundus. The distal part of the stomach, the antrum, merges into the pyloric canal, ending at the pyloric sphincter 188. The pars oesophagus is covered with the same type of squamous epithelium as the oesophagus and is not secreting mucus or enzymes 98. The cardia and the rest of the stomach, on the other hand, are covered with mucus secreting columnar epithelium. The cardia also contains mucus producing glands. The glands are composed almost entirely of mucus secreting cells. Few pepsinogen producing zymogenic or chief cells may be present. Gastric or fundic glands are located in the fundus, the greater part of the body and the proximal part of the antrum. The glands are lined with mucus producing cells, zymogenic cells, argentaffin cells which produce gastrin that stimulates release of gastric juice, and parietal cells. The latter produce hydrochloric acid and intrinsic factor for the uptake of vitamin B 12. Pyloric glands, which contain mucus producing cells and zymogenic cells, are located in the distal part of the antrum and pyloric canal. The mucus produced by glands and epithelial cells protects the underlying tissue from the corrosive mixture of HCl and pepsins. The lowest pH is measured in the distal part of the stomach, especially in the antrum, where the mixing takes place. The main function of HCl is considered to be the provision of a non-specific barrier against a variety of infectious organisms since luminal pH values of 1-2 can be detected 78, 121, 163. Emptying of the stomach into the duodenum is influenced by several facts (Table 2.1). Table 2.1 Factors influencing the emptying of the stomach 163. i Gastric motility. ii Volume, since the greater the volume of the gastric content the faster the emptying. iii Hypertonic contents are released slower than isotonic contents. iv The presence of fats or fatty acids and acid in the duodenum inhibits gastric emptying. v Cold liquids (4C) are emptied slower then warm liquids. 15 2.1.4 The small intestine In humans, the small intestine is 4-5 m in length. The duodenum makes up the first 25 cm, the jejunum refers to the next 2 m and the remainder constitutes the ileum. There is, however, no clear boundery between the regions. The digesta from the stomach pass into the duodenum where they are mixed with the secretions from the liver. The duodenum contains only one type of gland, the glands of Brunner that secrete mucus and bicarbonate. The release of bicarbonate is increased by the introduction of gastric juice in the duodenum. Bicarbonate is also released into the duodenum with pancreatic juice and secreted by the epithelial cells. Bicarbonate neutralises the digesta coming from the stomach and raises the pH to above 5. In the jejunum, the pH gradually rises to slightly above 6, in the mid ileum the pH becomes more neutral, pH 6.88 121. In addition to HCO3-, pancreatic juice also contains a mixture of enzymes, or their precursors, to hydrolyze carbohydrates, proteins and lipids. It also contains several enzymes capable of hydrolysing minor food constituents, e.g. nucleotides 163. Bile is excreted into the duodenum from the liver. The major organic constituents are bile acids, phospholipids and cholesterol which aid in lipid digestion, and bilirubin which gives faeces their characteristic colour. Major electrolytes in bile are K+, Na+, Cl- and HCO3-. One of the major functions of the small intestine is the absorption of nutrients. The small intestine is well adapted to this function. The surface area is enhanced some 600 times by morphological structures such as the villi and the circular Kerckring folds that are dominant in the jejunum. The villi are finger like structures that project into the intestinal lumen and are about 1 mm high. There are 10-40 villi per square millimeter. The luminal surface of the villi is lined with epithelial cells, referred to as enterocytes. The enterocytes further enlarge the available surface area since the membrane presented to the lumen is covered with microvillous structures approximately 1 m high and 0.1 m in diameter. The membrane is appropriately called the brush-border membrane 163. Several oligosaccharidases, peptidases and phosphatases are bound to the brushborder membrane, 115. These enzymes are expressed by mature enterocytes located towards the tip of the villi 76. The main functions of the enterocytes are digestion and the uptake of nutrients. Enterocytes are covered by a protective mucus gel (section 2.2) that is between 5 and 400 m thick 1, 22, 134. At the base of the villi are the crypts of Lieberkühn. These are simple tubular glands containing mostly undifferentiated cells as well as mucus secreting goblet cells, lysozyme and cryptdins producing Paneth cells 56 and endocrine cells. The undifferentiated cells divide and differentiate to 16 become the enterocytes lining the villus as they progress out of the crypt towards the villous tip, as on a conveyor belt. In humans, it takes about 6 days for cells to migrate from the base of the crypt to the tip. In many other mammals, this process takes from 1 to 3 days. Due to uptake of nutrients and water, the volume of the digesta is gradually reduced. Because of this reduction in volume, and fewer and slower peristaltic contractions, the movement of the digesta is slower in the ileum than in the upper part of the small intestine. For humans, the overall transit time in the small intestine is 4-6 hours 188. 2.1.5 The large intestine, colon and caecum In humans, the colon is about 1.5 m long, depending on the length of the person. It is devided into four regions; ascending, transverse, descending and sigmoid colon. The caecum is a blind pouch, connected to the proximal part of the ascending colon, adjacent to the ileo-caecal valve, which connects small and large intestine. The mucosa of the large intestine differs from the that in the small intestine, there are no villi and the crypts of Lieberkühn are deeper. The crypts do not contain Paneth cells, but have more goblet cells. A few goblet cells can also be found between the enterocytes. The motility of the colon is quite slow. A contraction wave slowely pushes the contents foreward at 1-2 cm.min.-1 78, 163. Because the contents do not flow through the colon as in a prop-flow reactor and because considerable back mixing takes place, especially in the ascending colon. A net forward movement of about 5 cm.h-1 is often noted 163, resulting in a transit times of up to 60 hours 188. 2.2 Mucus Mucus is lining the epithelium of the intestine. It is an important site for colonisation of both normal microflora and potential pathogens, and has been used as substratum for adhesion studies in papers II-V. 2.2.1 Function Intestinal mucus is a viscous substance produced by goblet cells and overlying the epithelial surface. The proposed functions of mucus are many (Table 2.2). 17 Table 2.2 i ii iii iv v vi vii Some of the proposed functions of mucus 22, 60. A barrier against chemical and proteolytic damage. Protecting the epithelium from mechanical damage. Lubrication, to facilitate the passage of the gut contents. Stabilising the micro-environment at the intestinal wall. Stabilising the association of some bacteria. Preventing the association of others. An energy source for the normal microflora. 2.2.2 Composition Mucus is a complex mixture that is predominantly water (95% or more). Large glycoproteins, mucins, are responsible for the viscous nature of mucus and make up 0.5-5%. Mucin monomers have a Mr of approximately 250 K and consist of a protein core that makes up some 20% by weight, and carbohydrate which constitutes some 80% by weight. Between 40-70% of the aminoacids are threonine, serine and proline. The central part of the protein is glycosylated and contains many tandem repeat sequences (each repeating unit being 6 to 169 amino acids in length, dependent on the particular mucin) 113, 126, 191. About 70% of the hydroxyl groups of serine or threonine are in O-glycosidic linkage with N-acetylgalactosamine. The oligosaccharide units are bound to this N-acetylgalactosamine and radiate out like the brisles of a bottle-brush 191. The dominant sugars are N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose and sialic acid. The number of monosaccharides linked to GalNAc rarely exceeds 18, and the chain may be branched. If the terminus of the chain consists of GalGlcNAc disaccharide, it carries a bloodgroup determinant 22, 21, 60, 126. Sialic acids give mucin its negative charge, pKa=2.6 123. The highly glycosylated part is relatively resistant to proteolysis. This part is flanked by less glycosylated, “naked”, regions. These regions are sensitive to proteolysis and may contain hydrophobic regions to which bacteria may bind, by non-specific hydrophobic interactions 21, 181. Two models exist for the polymeric structure of mucin. i) Four mucin monomers are bound to a central ‘link’ peptide, like the sails of a windmill, by disulphide bridges between the “naked” regions and the ‘link’ peptide 142. The size of the ‘link’ peptide has been suggested to be 70 K 142, 90 K 124 and 118 K 154, 156. The peptide makes up about 25% of the total mucin protein and 10% of the total mucin carbohydrate. Mannose constitutes 10% of the ‘link’ peptides carbohydrate 156. The existence of the ‘link’ peptide is very much debated, and according to some workers 179 it does not exist. 18 ii) The other model proposes a flexible thread structure, where mucin monomers are linked end-to-end via disulphide bridges. Electron microscopic results, favor the latter model. They show 0.2-4 m long threads which are neither branched nor star-shaped 155, 173. Lipids constitute up to 40% of the dry weight of intestinal mucus 178. About half of the lipids present in mucus are neutral lipids (free fatty acids, cholesterol, cholesterol esters, mono, di- and triglycerides). Glycolipids (glyceroglucolipids and glycosphingolipids, from exfoliated cells) constitute approximately 45%. The remaining lipids (about 5%) are phospholipids 178. The lipids in mucus affect bacterial adhesion by modifying the hydrophobic interactions between a bacterium and its substratum. Lipids, especially cholesterol esters, also influence the viscosity and elasticity of mucus 127. Free proteins make up 0.5-1% of the mucus. Part of these proteins are perfused plasma proteins, of which albumin, IgA and IgG are the most prevalent 45. Some proteins may originate from shedded enterocytes 35, 196. Other proteins are secreted, these include secretory IgA, lysozyme, lactoferrin and proteinase inhibitors 21, 45, 116. Extracellular matrix molecules, particularly fibronectin, have also been shown to be present in mucus 179. Salts constitute approximately 1% of mucus. Small amounts of free amino acids, carbohydrates and nucleotides that are most likely from lysed host cells in particular leucocytes, are also present 21, 45, 129. 2.3 The normal microflora The microbes characteristically found at different sites of the body of healthy subjects, are refered to as the normal microflora 188. They will colonise specific habitats of the body surfaces and contents, and stay there until they are displaced by organisms better adapted to occupy the habitat 66. In addition to these colonising microbes, various transient microbes can be observed. Since they just pass through the tract, unless some factor induces a change in the normal microflora whereby the transient microbe can establish, their influence on the intestinal ecosystem is variable. The normal microflora has also an important function blocking the colonisation by potential pathogens, more about that in section 4.2.1. 2.3.1 The mouth The mouth is one of the few places where the normal microflora is directly involved in physiological damage and disease. Due to the different tissues and great range in the redox-potential, a variety of habitats exists. The 19 normal microflora can adhere to many different substrata. Bacteria also adhere to each other (coaggregation), forming a bacterial mass known as plaque. Some of the most common genera found in the oral cavity are: Actinomyces, Lactobacillus, Streptococcus, Bacteroides, Prevotella, Fusobacterium, Neisseria and Veillonella. Yeasts of the genus Candida are also commonly found 136, 188. 2.3.2 The oesophagus Lactobacilli and other, unidentified, Gram-positive rods and cocci have been found to colonise the oesophagus of many animals 188. In humans, aerobic organisms were found to be present in all cases, while anaerobic organisms were present in 80% of the cases and yeasts in 12% 177. It has been suggested that the organism found in the oesophagus originate from the oropharynx and ingested food 71. 2.3.3 The stomach In humans, the stomach normally contains only small numbers of microorganisms. Up to 104 cfu.ml-1 gastric content has been reported 188, and many of these are in fact transient. They originate from food and the oropharynx 177. The microorganisms found are usually aciduric species; lactobacilli, streptococci and Candida albicans. A high percentage of people are colonised by Helicobacter pylori. Its natural habitat appears to be the mucus covered non-acid-secreting epithelium of the antrum. Many factors combine to induce H. pylori cells to change to become pathogenic after many years of being a commensal 110. In pigs, the secreting epithelium and especially the squamous epithelium of the pars oesophagus are colonised by lactic acid bacteria 38, 68. Desquamation of the pars oesophagus epithelium is supposed to inoculate the stomach contents with lactic acid bacteria, thus ensuring their dominance 7, 68, 98, 188. Unfortunately this theory has not been tested by surgical removal of the pars oesophagus. It could explain why animals that do not possess a pars oesophagus, for example humans, have no resident microflora in the stomach 121, 177 and lower numbers of lactobacilli in their intestine 188. 2.3.4 The small intestine In humans, the duodenum appears not to have a permanent microflora. The microflora is affected by the pH of the stomach and duodenum, also the 20 swift flow of the digesta reduces the chance for colonisation, 103-104 cfu.ml-1 content can be observed 177, 188. The composition of the normal microflora resembles that of the stomach 71. In the upper jejunum, up to 105 cfu.ml-1 and in the lower jejunum up to 107 cfu.ml-1 were observed by Macy and co-workers 121. Most of these microbes were anaerobes. Due to the slower passage of the digesta in the ileum, colonisation occurs, with bacterial populations of 107-108 cfu.ml-1 being observed 177, 188. The composition of the microflora in the ileum resembles that of the colon. Major constituents are lactobacilli, enterococci, members of the Enterobacteriaceae family, e.g. E. coli, and the obligate anaerobes, Bacteroides, Veillonella and Clostridium 121, 188. 2.3.5 The Large intestine Due to the slow flow rate, the colon is the main site of microbial colonisation in humans. About 1010-1011 bacteria per gram contents are present 163, 188. The number of bacteria increases one order of a magnitude from proximal colon to distal colon 119. Over 40 genera and 400 species have been isolated and identified from the faeces of individuals on several countries 166. Anaerobes out number aerobes by a factor 1000, due to the low oxidation reduction potential. It is interesting to note that some oxygen is available in the colon, because it diffuses from the epithelium and is present in the lumen where as much as 1.8% O 2 has been reported 163. The major genera found in the colon are: Bacteroides, Bifidobacterium, Eubacterium, Ruminococcus, Peptostreptococcus, Bacillus, Clostridium and Lactobacillus 163, 166, 188, 177. These bacteria have a fermentative metabolism in the large intestine. Dietary polymers, principally polysaccharides, and endogenous macromolecular polymers, mainly mucus, that reach the colon can be hydrolysed by the majority of the colonic bacteria and serve as an energy and carbon source. The major metabolic end products are short chain fatty acids. These fatty acids, especially butyric acid, are a major energy and carbon source for the enterocytes lining the colon. They provide an estimated 10% of the daily energy need in humans 165. 2.4 Enteric pathogens 2.4.1 General A pathogen can be defined as a disease producing organism 147. One should bear in mind, however, that any microorganism that can sustain itself in an other organism (e.g. human), may occasionally cause disease 21 and this will be influenced by the health status of the host 59. In order to be able to cause disease, a microorganism can possess so-called virulence factors. These are special properties which distinguish potential pathogens from harmless strains 93. Adhesion of pathogenic E. coli has been the main subject of study for this thesis. Its virulence factors will be discussed in more detail in the next section (2.4.2). Adhesion of other entropathogens has also been tested, but has usually been compared to the results obtained for E. coli strains. 2.4.2. E. coli Most pathogens are “one-disease” organisms, however, E. coli is able to induce a variety of diseases. Except for diarrhoea, many common diseases are caused by E. coli 93. E. coli is the most common cause of urinary tract infection 93 and neonatal sepsis 64. The E. coli strains that are able to cause disease, can possess many different virulence factors. They possess flagella (H-antigen), which make it motile, and fimbriae which allow it to adhere to host tissues and also confer host specificity (section 5.3). Fimbriae or fimbriae-mediated adhesion, is also thought to affect membrane structure and integral proteins, imparing water and electrolyte absorption and ATPase activity 19, 167, 168. Some E. coli strains can excrete enterotoxins in the intestine. Two different kinds exist; a heat labile toxin (LT) and a heat stable toxin (ST) 159. Two different types of LT exist. LT-I has a high degree (75%) of amino acid sequence identity with choleratoxin, and LT-II which so far has been found primarily in E. coli strains isolated from animals, has not been associated with disease. LT-I consists of five B subunits and one A subunit which is enzymatically cleaved in A1 and A2 subunits. Unlike V. cholerae, E. coli does not excrete LT-I. It possibly leaks out of the periplasmic space in the presence of bile acids and trypsin or in the absence of iron. The toxin activates adenylate cyclase which raises the cAMP level in the epithelial cell. Raised cAMP levels cause, among other things, excretion of Na + and K+ into the lumen. Water will follow the electrolytes due to osmotic activity 159, 162. ST is a family of toxins with a low molecular weight and contain 17 to 31 amino acids. They are compact and contain 3 disulphide bonds and are therefore not easily inactivated by heating. The heat stable toxins are subdivided into methanol soluble, ST-I (STa) and methanol insoluble, STII (STb) forms. ST-I activates guanylate cyclase which raises cGMP level. Two ST-I subfamilies exist: ST-Ia (STp), from bovine, porcine and human origin; and ST-Ib (STh) which is of human origin only 69. A rise in cGMP level leads to a similar loss of electrolytes and water as with raised 22 cAMP levels. ST-II functions differently from ST-I and has so far only been found in E. coli strains from porcine origin 159, 167. Other virulence factors are the capsule (K-antigen), lipopolysaccharide (endotoxin, O-antigen) and haemolysins. Capsules loosely coat the bacterial cell and are composed mainly of carbohydrate with some aminoacid or lipid components. Capsules protect the bacterium from the hosts defence mechanisms, complement activation and phagocyte mediated killing 93, 159. Almost without exception, SfaII expressing E. coli cells possess a K1 capsule which is a sialic acid polymer 104. Sialic acids do not trigger an antibody response because they are also present on the hosts cells 159. LPS makes up the outside of the Gram-negative outer membrane. It consists of core polysaccharide, the O side chain and lipid A which is the hydrophobic part that anchors LPS in the membrane. LPS is thought to protect the bacterium against the hosts complement system 93, 147. It is also responsible for many pathophysiological effects; inflammation, blood coagulation, fibrinolysis and hypotension 147. Two types of haemolysin can be produced by E. coli; secreted haemolysin and cell bound -haemolysin. Haemolysins form pores in the membranes of cells, not only erythrocytes. It is thought that the haemolysin releases iron from erythrocytes, disrupts phagocyte function and is directly toxic to host tissues 93. 23 3 Milk One of the characteristics of mammals is the production of milk with which they suckle their young. Milk not only provides nutrition, but also a range of factors that protect the newborn from many enteric pathogens. Of the 4000 or so mammalian species, the milk of only 5 or 6 species are used by man. In addition to human milk, bovine milk is most widely used 62. 3.1 Composition and functions Milk can easily be devided in four fractions, namely, fat, cells, casein and whey. Milk whey has been studied in this thesis. Fat and cells can be separated from the milk by centrifugation. The cells which include macrophages, neutrophiles, lymphocytes 188 and cell debris will form a pellet while the fats will collect at the top. Milk fat is concentrated in globules with a diameter of 1-10 m. The globules are surrounded by a membrane, rich in mucins 169, 175. These membranes will also collect in the top layer. Casein can be separated in two ways. It can be precipitated by lowering the pH to about 4.6 which is the isoelectric point of casein. Alternatively, it can be coagulated by hydrolysis of -casein with proteases. Whey, is the solution left after removal of casein 130. Whey prepared according to the former method was used in paper IV, and whey prepared according to the latter method was used in paper V. Minor differences in the composition of both types of whey exist. Lactose makes up around 70% of the dry matter in the whey from cow milk. Only 11-12% of whey from cow milk is protein 128. 3.2 Milk whey 3.2.1 Oligosaccharides The main sugar present in both human and cow milk is lactose. The concentration in human milk (55-70 g.l-1) is about one and a half times higher than in cow milk. Human milk also contains a wide range of other oligosaccharides (3-6 g.l-1), while only traces are detected in cow milk 107. N-acetylglucosamine (GlcNAc) containing oligosaccharides have been shown to act as “growth factors” for esp. Bifidobacterium bifidum var. pennsylvanicus 144. This sugar is rather rare in cow milk. This could explain the some times observed difference in the number of bifidobacteria in the faeces of breast fed v.s. formula fed infants. Formulas often contain transgalactosylated oligosaccharides. Fermentation of these 24 oligosaccharides or GlcNAc in the large intestine, lowers the pH and creates an unfavourable environment for a number of pathogens. 107, 188. Many of the oligosaccharides naturally occurring in human milk are thought to function as soluble receptors for pathogenic bacteria and viruses (see chapter 6 on inhibition of adhesion). 3.2.2 Proteins In bovine milk, whey proteins represent 20-25% of the total milk proteins. The major whey proteins are -lactoglobulin, -lactalbumin, immunoglobulins and blood serum albumin. Minor proteins are: lactoferrin, lysozyme, lactoperoxidase, 2-microglobulin, free secretory component etc. 62 a) -lactoglobulin -lactoglobulin (-lg) represents approximately 50% of the whey proteins in cow milk. Human milk does not contain -lg. Bovine -lg originating from the diet can be observed in human milk 90. The monomeric molecular weight of -lg is approximately 18 000. In milk, -lg normally exists as a dimer. It is proposed that the biological function of -lactoglobulin (-lg) is that it binds retinol, thus protecting it against enzymatic oxidation and facilitating its absorption. If that is in fact the physiological function of -lg, one must question how retinol is protected and how efficiently it is absorbed from human and rodent milk that lack this protein 62. At present, attention is being focused on the fact that -lg may be one of the causes of cow milk allergy 63. In paper IV and V, it is shown that -lg also appears to have antimicrobial activity, because it inhibits S-fimbriae mediated adhesion, see also section 6.3.6. b) -lactalbumin -lactalbumin (-la) is the next most prevalent whey protein and constitutes 20% of all whey proteins in cow milk. In human milk there is considerably more since -lactalbumin is part of the lactose synthetase complex. It modifies the action of -galactosyltransferase so that lacose is produced. The complex catalyses the addition of galactose to glucose to produce lactose. -la contains a Ca2+-ion which could provide protection against thermal denaturation 185. Although removal of the Ca2+-ion results in denaturation of -la at approximately 50C 62, this is not likely to be a biological function because milk will not be exposed to such 25 temperatures under natural conditions. It is more likely Ca2+ functions as a second messenger, regulating the catalytic properties of the lactose synthetase complex 185. -la has also been shown to be a growth promoter for certain bifidobacteria 144. c) Immunoglobulins Bovine colostrum contains up to 100 g.l-1 immunoglobulins, but the concentration decreases to less then 1 g.l -1 within a week. Human milk contains approximately 10 g.l-1 immunoglobulins after two days, this decreases to some 2 g.l-1 after two weeks 62. Three classes of immunoglobulins are found in milk; IgA (the main immunoglobulin in human milk), IgG (the main immunoglobulin in cow milk) and IgM. IgG is monomeric, IgA dimeric and IgM pentameric. The monomers consist of four polypeptide chains, two heavy (Mr 50-70 K) and two light (Mr 25 K), linked by disulphide bridges. The di- and pentamers are made by linkage of the monomers with a protein called the J-chain. IgA contains a secretory component, which facilitates the transport of IgA from the blood to the extracellular side 62, 184. The role of the immunoglobulins function is to provide passive immunization for the newborn, mainly by IgA and IgG 52. An entero-mammary circulation has been described, in which antibody-producing cells from the maternal intestine migrate to the mammary glands. They produce secretory antibodies against enteric pathogens common in the maternal-newborn environment and are provided to the newborn in the milk 46. The presence of anti-idiotypic antibodies, antibodies that “look” like the antigen, in human milk has been suggested. This might result in even an active immunization of the newborn 83. d) Serum albumin Both human and bovine milk contain around 0.4 g.l -1 serum albumin. Bovine serum albumin has a calculated molecular weight of 66 267 Da, it has an overall ellipsoidal shape, with an axial ratio of about 1:3. In the circulation, serum albumin transports insoluble hydrophobic molecules like bilirubin and fatty acids, possibly in hydrophobic pockets that can open and close 52. Cations, especially Cu2+ and Ni2+ can be bound on the surface 62. No specific function for serum albumin in milk has been found thus far, although its fatty acid binding ability may aid lipolysis. Serum albumin concentrations in blood are some 10 times higher than in milk. It may leak out of the circulation into the milk 62. 26 e) Minor whey proteins Most of the previously mentioned minor proteins appear to have some antimicrobial activity. Lactoferrin is a glycoprotein with a molecular weight of about 80 000. It can bind irreversibly two Fe3+ per molecule. This iron binding activity has been reported to form the basis for lactoferrins antimicrobial activity 52, 62 (see section 6.2.1). It removes the iron necessary for growth of most bacteria, with the exception of lactic acid bacteria. The highly cationic nature of lactoferrin could be responsible for another antimicrobial effect of lactoferrin, namely damage of the outer membrane of Gram-negative bacteria 117, 199. Cow milk contains 0.02-0.35 mg.ml-1 lactoferrin and this is 10 to 100 times lower than the concentration in human milk 62. The antimicrobial activity lysozyme has been discussed in section 2.1.1 for lysozyme in saliva. Lactoperoxidase is a glycoprotein with a Mr of approximately 77 500. It is a mammalian peroxidase found in saliva, tears and milk. It catalyzes the oxidation of thiocyanate (SCN-) by hydrogen peroxide, generating OSCNand possibly also HO2SCN and H3OSCN. Lactoperoxidase makes up about 1% of whey protein. The substrates, thiocyanate and H2O2 are derived from glucosinolates and cyanogenic glucosides, and produced by lactic acid bacteria 70, 148. Structural damage and changes in bacterial membranes due to exposure to OSCN- have been reported 100. However, the main bacteriostatic effect is the contribution to blocking of the glycolysis. It is proposed that it inhibits glucose transport, hexokinase activity and glyceraldehyde 3-phosphate dehydrogenase activity due to oxidation of sulfhydryl groups in metabolic enzymes. The latter enzyme appears to be the primary target 20. 2-microglobulin (lactolin) has a Mr of 12 000 and is structurally similar to immunoglobulins 62. It is thought to stabilize the tertiary structure of histocompatibility antigens or is necessary for processing and intracellular transport of the antigen 79. Secretory component is a glycoprotein with a molecular weight of 74 000. It not only acts as a receptor for IgA to be secreted but has also been found to inhibit the adhesion of enterotoxigenic E. coli 74 (see section 6.2.1). 27 4 Lactobacilli 4.1 General background The genus Lactobacillus includes regular, non-sporing and Gram-positive non-motile rods. Since lactobacilli are unable to synthesize porphyrins, they usually lack catalase and cytochromes, unless the porphyrins are present in the growth medium. They are “aerotolerant” anaerobes, are fastidious and have a fermentative metabolism, producing lactic acid as the major metabolite during sugar fermentation. Lactobacilli are aerotolerant and grow optimally under slightly acid conditions 6, 147. Although their name suggests that lactobacilli are associated with milk, they can be found in a wide range of other nutrient rich environments such as plant matter and meat as well as being part of the normal microflora of the mouth, intestine and vagina of mammals 6, 147. Although lactobacilli are members of the normal microflora of the intestine (section 2.3), it should be noted that they are not numerically dominant, and that approximately 20-25% of humans do not have lactobacilli in their intestine 166, 188. 4.2 Proposed functions in the intestine At the turn of the century, Metchnikoff suggested that oral ingestion of beneficial bacteria could interfere with harmful bacteria. He regarded the normal microflora of the large intestine as extremely harmful 188, 187. Today it is aknowledged that the normal microflora performs an important protective role against disease (see section 4.2.1). Lactobacilli are thought to improve the stability of the intestinal microflora, thereby having a beneficial effect on the health of the host 157. This use of microorganisms, is also called probiosis. The term probiotic has been defined as a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance 66, although it is used today as food supplements as well. Many beneficial properties have been proposed for probiotics and lactobacilli in particular. Some of these are listed in Table 4.1. In order to exert one or more of these beneficial effects, the probiotic strain has to be present at the site where it is required. It would therefore be desirable for the strain to colonise the host at that site. That could prove to be difficult since in a host all possible colonisation sites could already be occupied by members of the normal microflora or established pathogens. The probiotic strain should have to replace a member of the normal microflora, which is highly unlikely (see next section; 4.2.1) 164. In a host with a disturbed microflora, this might be more feasaible. If the 28 probiotic strain can not colonise, it may have to perform while “passing through”. In the upper gastro intestinal tract, this may work due to the lower numbers of the normal microflora 158. Table 4.1 Some of the proposed beneficial properties of probiotics 40, 67, 84, 137, 161. Property Proposed mechanism Anti carcinogenic activity stimulation of the immunesystem, inactivation of mutanogens and carcinogens, reduction of enzymes implicated in carcinogen production. Prevention of deconjugation of bile acids. hypercholesterolaemia Improved nutritional status of vitamin production, predigestion of antifoods nutritional factors, metabolism of lactose. Stimulation of the immune lactobacillal cell walls act as adjuvants. system Alleviation of constipation improved intestinal motility. Prevention of osteoporosis Prevention of diarrhoea Improved growth rate of farm animals increased bioavailability of minerals. stabilising the normal intestinal production of antimicrobials, blocking of adhesion sites, inactivation of enterotoxins, colonisation resistance (see also section 4.2.1). Suppression of sub clinical infection by growth depressing organism. 4.2.1. Colonisation resistance Lactobacilli and other microorganisms may prevent diarrhoea by producing components that are antagonistic towards pathogenic bacteria. Many mechanisms are involved in this antagonism. The normal microflora is considered to have a protective function against disease. This was demonstrated by the finding that treatment of mice and guinea pigs with antibiotics eliminated part of the normal microflora. This loss made them extremely sensitive to infection by Salmonella enteritidis and Vibrio cholerae (ID50, 106 bacteria for untreated animals v.s. less then ten bacteria for antibiotic treated animals) 85. Even more convincing was the finding that germ-free guinea pigs could be killed by as little as ten S. enteritidis cells, while it required 109 cells to kill an animal with a normal microflora 36. 29 The protection provided by the normal microflora is referred to by different terms: Environmental resistance, colonisation resistance, premonition, infection immunity, antagonism, bacterial interference, microbial interference or competitive exclusion [187]. In this thesis, I will use the term colonisation resistance. In the intestine, microorganisms are competing with each other and in some cases with the host. For the latter situation there is competition for nutrients, not only carbon sources but also for scarce nutrients such as iron [187]. Microorganisms are competing with each other for sites, including sites on the mucosal surfaces [85]. Microorganisms can more successfully compete by producing antimicrobials, either as metabolites (e.g. volatile fatty acids, hydrogen peroxide etc.) or by the production of compounds refered to as bacteriocins. Some strains of lactic acid bacteria have been found to produce bacteriocins. Klaenhammer 101 defined four distinct classes of bacteriocins from lactic acid bacteria. The typical bacteriocin, however, is a small amphiphilic protein with antimicrobial activity that is generally specific for closely related strains 91. This is also a potential disadvantage as they might work against other beneficial strains instead of against unrelated pathogens 161. Bacteriocin producing Lactobacillus strains can be isolated from faeces. It has also been shown that E. coli cells that produce a bacteriocin referred to as colicin, can colonise volunteers better and longer than non-colicinogenic E. coli cells 163. It is, however, not known whether bacteriocin production is important physiologically [187, 188]. To answer this question, one could use a method similar to that used by Blom and Mörtvedt 13 for the demonstration of bacteriocin production in sausage. These workers used a bacteriocin producing strain together with an isogenic non-bacteriocin producing, bacteriocin sensitive strain. In paper I it is shown that the growth inhibiting activity of acidic metabolites from L. fermentum 104 can be enhanced by components with an Mr over 1000. Spent culture fluid from L. fermentum 104 was bactericidal to ampicillin resistant E. coli strain 1107. This activity could not be inactivated by heat or protease treatment, but was found to be pH dependent (Fig. 5 paper I). These results are typical findings for growth inhibition by short chain fatty acids 161. Spent culture fluid fractions with molecular weights less than 500 and 1000 were, at best, bacteriostatic (Fig. 1 and 2 paper I), however, fractions with molecular weights more than 500 or 1000 did not show bactericidal activity. These findings suggest the presence of high a molecular weight component which is not bactericidal by it self, but which may enhance the activity of the acidic metabolites present in the spent culture liquid or be activated by them. This hypothesis is strengthened by the observation that casaminoacids restore the bactericidal activity of the <500 fraction. Enhanced bactericidal activity of 30 organic acids has been observed before. Milk and lysed horse blood were found to greatly enhance the bactericidal activity of acetic acid against Salmonella sp. 31. 31 5 Adhesion 5.1 General background Adhesion to a surface is important to bacteria in most environments. It enables them to colonise environments under conditions where they otherwise would be washed away 201. For example in the small intestine the flow is rather high due to secretion of fluids into the lumen, and peristaltic movements (1-2 cm.s-1163). In humans, transit times of 4-6 hours have been reported 188. In addition, epithelial cells with attached bacteria are shed, and mucus is released into the lumen continuously. Adhesion to the intestinal mucosa is regarded as a prerequisite for colonisation of the small bowel by both pathogens and the indigenous microflora. 5.2 Non-specific adhesion Non-specific interactions were defined by Busscher and Weerkamp 18 as interactions due to overall macroscopic surface properties, as charge or surface free energy. Non-specific adhesion is the most common form of adhesion in nature. It involves non-covalent bonds and hydrophobic interactions. Non-covalent bonds are: i Electrostatic forces, which are weak in water due to its high relative permittivity. Multivalent cations have been found to enhance adhesion 30, 17. They can form a bridge between the bacterium and the host cell by electrostatic interaction to both. ii Hydrogen bonds are also weakened by the presence of water. iii Finally, van der Waals forces. This type of bonding works at distances of over 50 nm, but has a sharp maximum at approximately 0.5 nm 184. Due to plasma membrane phospholipids, teichoic acids, lipopolysaccharides, acidic sugar residues and other factors, the net surface charge of both bacteria and host epithelial cells is negative, thus causing a repulsion between the cells. The so-called DLVO theory 50, 194 describes the interaction between particles of the non-covalent forces (see Fig. 5.1). It predicts that two surfaces of similar charge may attract each other by long-range (>50 nm) forces, created by fluctuating dipoles. At 1020 nm there exists an energy minimum. Adhesion in this so-called secondary energy minimum is rather weak and reversible. At shorter distances, the repulsive forces become stronger due to the overlap of the diffuse electrical double layer (adsorbed ions on the surfaces) of both particles. The electrostatic repulsion is strongly influenced by the ionic 32 strength of the solution (that is the ion valence and the concentration). The higher the ionic strength, the thinner the electrical double layer and this results in less repulsion. When the repulsive forces are overcome, the attractive forces become stronger again and create a primary energy minimum. The forces in this primary minimum are stronger than in the secondary minimum and binding is irreversible. Kinetic energy is required to overcome the electrostatic repulsion of the diffuse electrical double layer between both minima, however, under normal conditions, bacterial cells do not have enough kinetic energy 95. Fimbriae (discussed in section 5.3) on the bacterial surface are much thinner (3-10 nm) than the bacterium itself (0.5-1.5 m). They have a smaller radius of curvature and therefore have less surface that is parallel with the substratum. Therefore, they experience less repulsion. The fimbriae can overcome the electrostatic repulsion and span the distance between the primary and secondary minimum. They can bind in the primary energy minimum, often by more specific interactions, thereby anchoring the bacterium firmly and irreversibly to the host tissue. Enteropathogenic E. coli appear to bind directly in the primary minimum, possibly by inducing changes in the brush border membrane, rather than by fimbrial attachment 53. Figure 5.1 Attractive and repulsive forces working upon a particle (bacterium) approaching a solid surface, according to the DLVO theory 50, 194. Hydrophobic interaction is mainly entropy-based. Water molecules around a hydrophobic surface are more ordered then around a hydrophilic one as they can not make many hydrogen bonds at a hydrophobic surface. 33 Interactions between two hydrophobic surfaces in an aqueous solution, therefore liberates the ordered water molecules originally surrounding the surfaces. This increases the entropy and reduces the free energy of the system 28. Craven and co-workers 43 found that non-specific binding, especially hydrophobic interaction, plays an important role in in vitro binding of Salmonella sp. to chicken mucus and enterocytes. Adhesion of Streptococcus sobrinus and Streptococcus sanguis to saliva coated hydroxyapatite has also been found to be non-specific 133. Hydrophobic interaction was found to play an important role in the adhesion of marine yeasts irrespective of the hydrophobicity of the substratum 193. Adhesion of E. coli HB101 to HeLa cells was also found to be dependent on surface hydrophobicity of the bacteria 41, as does the adhesion of Yersinia enterocolitica to brush border membranes 150. Breines and Burnham 16 reported that they could reduce non-specific adhesion to uro-epithelial cells, by treating the cells with 0.4 M HCl. In fact, this treatment would hydrolyze mucus normally covering the cells and thereby reduce specific adhesion to the mucus rather than reducing non-specific adhesion as proposed. In table 4 (paper III) it can be seen that many of the tested strains bind as well to BSA as to porcine ileal mucus. The observed adhesion to BSA is regarded as non-specific adhesion. Infact, BSA is generally used as blocking agent in binding studies, e.g. 15, 75, 109. In this thesis, the term non-specific adhesion is used to refer to adhesion which is not a specific receptor-adhesin interaction, as described below. Although non-specific, in the in vivo situation, non-specific adhesion may play a more important role than generally believed. 5.3 Specific adhesion After the bacterium has bound rather loosely to the host cell surface by non-specific interactions, specific interactions may develop 9. Specific adhesion typically involves a lock-and-key bond between complementary receptor and adhesin molecules. An adhesin is a structure on the surface of a microorganism, and a receptor is a complementary structure on the surface of a host cell 9. The adhesin molecule has to be presented in such a way that it can easily interact with the receptor and not be affected by interfering molecular structures and negatively charged molecules present on the bacterial surface 96. Many members of the Enterobacteriaceae family express fimbriae. Fimbriae had been observed by several workers, but some thought them to be artifacts. Houwink and van Iterson 87 were the first to suggest that they might be involved in the adhesive ability of certain 34 strains. Duguid and co-workers then showed that these structures were involved in haemagglutination and adhesion, and suggested their biological function to be the binding to cells and solid particles in the intestine 54. Fimbriae were shown to be fibre like structures (hence their name) made of polymers of polypeptide units, arranged around a central canal. Each bacterial cell possess several hundreds of fimbriae, which are spread peritrichously over the cell and are anchored to the outer membrane. They are approximately 0.3 to 2.0 m long and 3 to 10 nm thick 55, 93. The adhesive molecule can be situated on the tip of the fimbria, as with P fimbriae and CFA/I 17, 49, 96, both on the tip and laterally, as with type 1 fimbriae 102, or laterally as with K88 and K99 fimbriae 49. The receptors proposed for the fimbrial adhesins that were used in this thesis are presented in table 5.1. Except for K. oxytoca type 3 fimbriae, all adhesins bind to carbohydrate moieties. Carbohydrate-binding proteins usually contain a hydrophobic cleft with aromatic and charged residues projecting into it. The former interact with the carbohydrate backbone and the latter with the hydroxylgroups of the carbohydrate 106. This has been found to be true for fimbriae too. Many fimbriae are hydrophobic 49 or possess hydrophobic pockets, like type 1 fimbriae 93, 102 and P fimbriae 93. In addition, P fimbriae make five hydrogen bonds with a polar ridge of Gal(1-4)Gal 106. So, the same non-covalent bonds which play such an important role in non-specific binding, are also responsible for the interactions in specific binding although at a much shorter range and therefore requiring the right stereochemical conformation to guarantee optimized fit 138. Even though the binding of each fimbria is rather weak, a bacterium possesses several hundreds of fimbriae which together attach the bacterium firmly to the substratum. Binding of enteropathogens to carbohydrate moieties is not surprising. Epithelial cells lining the intestinal mucosa have a glycocalyx on the surface. This glycocalyx provides a surface rich in glycoproteins and glycolipids. Glycolipids and especially glycoproteins present in mucus and/or on epithelial cell membranes, are considered to be receptors for pathogens. For many types of fimbriae, several glycoprotein receptors have been isolated. It is not unlikely that one adhesin has many different receptors, however, it could also reflect differences in affinity by different fimbrial serotypes, differences in glycoprotein from porcine and murine origin or from erythrocytes 145, and possible proteolytic degradation prior to isolation 49, 96. 35 Table 5.1 Fimbrial adhesins discussed in this thesis and their proposed receptors. Bacterium/Fimbrium Receptora Escherichia coli K88 -D-Gal, fucose58, GalNac, GlcNac3, Gal(1-3)Gal 197, Galactosylceramide15 E. coli K99 GalNac(1-4)Gal(1-4)GlcCer, NeuGc(23)Gal(1-4)Glc(1-1)Cer140, 180, 189 E. coli F41 GalNac, GlcNac114 E. coli 987P Fucose, Glucose, Galactose, Mannose, corresponding amino sugars and N-acetylated derivatives 48 E. coli, Salmonella Mannosides typhimurium Type I E. coli CFA GalNac(1-4)Gal(1-4)GlcCer, Gm2 ganglioside, sialoglycoprotein 145 E. coli Sfa NeuAc(2-3)Gal(1-3)GalNAc103, 141 Yersinia enterocolitica Mucin carbohydrate moiety125, extracellular Yad A matrix proteins S. typhimurium Gal(1-3)GalNAc73 Salmonella enteritidis Fibronectin 37, plasminogen 176 thin aggregative fimbriae Klebsiella oxytoca non-carbohydrate segments of type V type 3 fimbriae collagen32 a Gal = Galactose; GalNAc = N-acetyl galactosamine; Glc = Glucose; NeuGc = N-glycolyl neuraminic acid; NeuAc = N-acetyl neuraminic acid; Cer = Ceramide; GlcNAc = N-acetyl glucosamine Recognition of different receptors by different adhesins allows bacteria to bind to specific tissues, e.g. urinary tract epithelium, different epithelial cells in the gastrointestinal tract 8, dental surfaces, other bacteria (coaggregation) etc. It also allows host specificity, e.g. E. coli CFA/I and CFA/II infections are limited to humans, E. coli K88 fimbriated cells infect pigs, E. coli K99 infections are limited to calves, lambs and piglets 49. The K99 fimbriae are less host specific. 5.4 Adhesion assays In vitro adhesion assays are used to investigate the adhesion of bacteria to various substrata. Haemagglutination was the first assay used to study the adhesive properties of fimbriated bacteria 54, 55. Subsequently, tissue 36 pieces 97, tissue culture cells 47, epithelial cells 43, 198, brush border membranes 172, immobilised mucus 35, 108, 120 and immobilised ileostomy glycoproteins paper III, IV and V have been used as substrata for adhesion. In principle, the substrata and bacteria are incubated together to allow binding to occur. Unattached cells are removed by washings, centrifugation or buoyancy 75. The number of bound bacteria can be determined by microscopy 47, colony forming units 183, radiometry 108, spectrophotometry 120, ELISA 160 and other enzyme linked assays 75. In this thesis, the Caco-2 cell line, immobilised porcine ileal mucus and immobilised ileostomy glycoproteins were used as substrata for adhesion. Human ileostomy glycoproteins that were a generous gift from Dr. J.G.H. Ruseler-van Embden (Erasmus University, Rotterdam, The Netherlands), were used as a model for human intestinal mucus. Adhesion of bacteria was determined radiometrically 35, 108. In short, mucus or ileostomy glycoproteins were passively immobilised by overnight incubation at 4C to polystyrene microtitrewells. Before use, wells were washed to remove excess mucus that was not immobilised. The Caco-2 cell line was grown under standard conditions and used after two weeks, when the cells were well differentiated. Caco-2 cells differentiate in vitro and express several properties that are characteristic for mature enterocytes. In order to study components that inhibit adhesion, the substrata were pre-incubated with test solutions, namely Lactobacillus spent culture fluid, bovine colostrum whey or their fractions. As controls the substrates were pre-incubated with uninoculated medium or buffer phosphate buffered saline (PBS), HEPES buffered Hanks salt solution (HH-buffer). After 1 hour at 37C the wells were washed and a suspension of radiolabelled bacteria was added. After incubation at 37C for 1 hour, the substrates were washed to remove unbound bacteria. Bound bacteria were released and lysed by incubation at 60C with SDS and NaOH. Activity was determined by liquid scintillation. The extend of inhibition of adhesion was determined by comparison of radioactivity in wells treated with test solution as compared to the radioactivity in wells treated with buffer or uninoculated medium as the control. 37 6 Inhibition of adhesion 6.1 General background As stated in the previous chapter, adhesion can play an important role in colonisation of the small intestine by microbes. Adhesion of a pathogen to host tissue is one of the first events in the development of many infectious diseases and is considered a prerequisite for infection 9, 59, 93, 152. Consequently blocking the initial attachment could effectively interfere with bacterial infection at an early stage 59. Adhesion could be inhibited by either affecting the adhesin on the bacterial cell or the receptor on the substrate. Phagocytes also carry receptors on their surface which allow them to recognize and bind to a variety of bacteria. Alternatively, bacteria can be opsonised with complement components and immunoglobulins, which subsequently bind to phagocyte receptors 153. After this binding, the bacteria are readilly phagocytosed and killed as part of the host defence mechanism 147, 89. It could be undesirable to interfere with this adhesion, except in the case of HIV 135. HIV binds to the CD4 receptor of T4 lymphocytes where after it infects the cell. 6.2 Affecting the adhesin. 6.2.1 Blocking the adhesin. The adhesion to the substratum can be influenced by the presence of soluble receptor analogues. These analogues will bind to the adhesin and block its binding site, making it impossible to bind to the real receptor on the substrata. The adhesive capacity of the bacteria will thereby be impaired. Intestinal mucus 65, 120, 156, Tamm-Horsfall protein (uromucoid or urinary slime) 93 and urinary oligosaccharides 99 are proposed to prevent in situ binding by functioning as natural receptor analogues. They block the binding site of the adhesin and potential pathogens would then be unable to bind to the underlying epithelium. Carbohydrate components that function as receptor analogues have also been tested in vivo and it has been proposed that this would reduce the concentration of K88 fimbriated E. coli 186. In this study, even though the E. coli K88 numbers decreased, the concentration of total E. coli was also lower in the test group, while the ratio of K88 fimbriated E. coli to total E. coli was unchanged. This suggests that a mechanism other than blocking of the adhesin by the carbohydrate receptor analogue may have been involved. 38 Cranberry and blueberry juice have been found to inhibit the adhesion of type 1 and P fimbriated E. coli to eukaryotic cells 200. Using an in vitro adhesion assay it was concluded that the active components were condensed tannins. The components bind to the bacterial surface, possibly to the adhesin itself 139. Clinical studies indicated that cranberry juice significantly reduced the incidence of urinary tract infections 5. It remains to be determined if the condensed tannins that were found to be the active component in the in vitro assay, are responsible for the in vivo effect. Sanchez and co-workers 160 found that a range of glycoproteins were able to inhibit adhesion of F17 fimbriated E. coli to bovine mucus and brush-border membranes. The glycoconjugates were proposed to work as receptor analogues, however, no proof was given that the glycoconjugates did not affect the immobilised mucus or brush-borders. Although the bacteria were preincubated with the glycoconjugates, the mixture was incubated with the immobilised mucus or brush-borders in the presence of the glycoconjugate. Milk has been found to contain substances that inhibit the adhesion of pathogens. These include immunologic components, glycoproteins and oligosaccharides. In one study 42 it was shown that oligosaccharides that inhibited adhesion of a urinary tract pathogenic E. coli were also detected in urine from both mother and the infant. Similar oligosaccharides were also present in the breast milk. As immunoglobulins provided in the milk are usually directed against a pathogen or its components, immunoglobulins affecting the adhesion of pathogens, will usually affect the adhesin. Fimbriae are strongly immunogenic antigens. Vaccination of dams with K88 antigen induced the presence of K88 specific IgG in the colostrum. The colostrum inhibited markedly the adhesion of K88 mediated adhesion to porcine intestinal tissue, however, levels fell progressively in the milk at 2 and 7 days postpartum. High levels of K88 specific IgG were also found in the serum, suggesting that vaccination with purified adhesin may be an option for inhibiting pathogen adhesion 97. Vaccination of pregnant sows with purified 987P fimbriae, gave high protection to piglets upon challenge with enterotoxigenic E. coli expressing homologous 987P fimbriae. In the control groups, 88.6% of the piglets developed diarrhoea v.s. 18% in the vaccinated group 143. A long term disadvantage of vaccination may be the selection for new fimbrial types 195. Cravioto and co-workers 44 reported that secretory IgA from human milk bound to an enteropathogenic E. coli adherence factor, thereby inhibiting its adhesion in a tissue culture cells. Free secretory component (see section 3.2.2e), from human milk, has been found to inhibit haemaglutination of CFA/I fimbriated E. coli 74. It was suggested that the oligosaccharide residues of free secretory 39 component may inhibit E. coli adhesion. Some of the saccharides found on free secretory component are galactose, mannose, fucose, glucosamine and sialic acid 149. There are reports that these components can function as receptors for many fimbrial adhesins (see Tab. 5.1). Numerous non-immunological factors have been found to block adhesion of enteropathogens. In many cases the activity has glycoproteins and oligosaccharides, which may act as soluble receptor analogues. S-fimbriae mediated adhesion was found to be inhibited by mucins in human milk 169, 170, 171. It should be noted that these mucins differ considerably from intestinal mucins 154. Unidentified glycoproteins in human milk have been proposed to work as soluble receptor analogues against E. coli 4, 86, V. cholerae 86 and Y. enterocolitica 92. Human milk contains a large quantity and a great variety of free oligosaccharides (see section 3.2.1). Many of these oligosaccharides have been identified as receptors for pathogenic microorganisms 44, 107. Lactoferrin has antimicrobial activity due to its iron sequestering activity, and inhibits the invasion of HeLa cells by E. coli expressing the Yersinia pseudotuberculosis invasin gene. Although lactoferrin was found to bind to the tissue culture cells, it was concluded that the binding to E. coli was responsible for the activity by disrupting the outer-membrane 116, 117, 199. It has also been suggested that oligosaccharide residues of lactoferrin were responsible for blocking of E. coli CFA/I binding 74 and type 1 mediated adhesion 190. Finally, a non-specific blocking of adhesion has been reported using bovine casein 133. It was proposed that the casein coated the S. sobrinus and S. sanguis cells, thereby changing the surface properties of the bacteria and inhibiting the adhesive capacity. 6.2.2 Reducing adhesin expression. A more direct way of influencing the adhesin, is reducing its expression. This has been shown to be possible for both Gram-positive and Gramnegative bacteria by using sub-lethal concentrations of antibiotics. Although antibiotics can affect slime production by bacteria and induce changes in the bacterial peptidoglycan structure, it is suggested that only the latter changes would affect the adhesive capacities 24. Changes in the peptidoglycan could induce changes in surface hydrophobicity and charge 118, 182. Exposure to sublethal concentrations of antibiotics can also influence fimbriae. Electron microscopy has been used to show that cells were partially or totally devoid of fimbriae after exposure to antibiotics 51. Subsequently, Breines and Burnham 16 showed presence of fimbriae, on 40 bacteria exposed to antibiotics, with the help of monoclonal antibodies against fimbriae. They argued that this inconsistency can be explained by the presence of fimbrial stumps, or by the formation of fragile fimbriae which can have broken off during preparation for electron microscopy. On the other hand, Eisenstein and co-workers 57 showed that significantly longer fimbriae are present on the bacteria, suggesting that the fimbriae are not particularly fragile. These workers suggested that fimbriae may be produced that lack an adhesin. Conway and Adams 39 showed that the food color erythrosine was able to inhibit adhesion of Lactobacillus sp. They concluded that erythrosine binds to the bacterial cell surface and alters bacterial metabolism, thereby preventing production of bacterial adhesin. 6.3 Affecting the receptor While affecting pathogen adhesion by affecting the receptor seems desirable, one must also consider that in so doing, one may interfere with another function of the receptor molecule since it is likely to be involved in biological processes, which may be disturbed. 6.3.1 No receptor. Specific adhesion, mediated by adhesins, requires the presence of a receptor. Presence or absence of a receptor may determine susceptibility or resistance, respectively, to a particular pathogen. As stated above, E. coli K88 infections are limited to pigs, however, not all pigs are susceptible to colonisation by E. coli K88. Two phenotypes of porcine enterocyte brush-borders have been identified, namely brush-borders that carry a receptor for K88 fimbriae and those that do not. The presence of the receptor was shown to be the dominant trait while absence of the K88 receptor was a recessive trait. At least 4 porcine phenotypes have been demonstrated since they bind K88ab and/or K88ac and/or K88ad fimbriae 12, 172. 6.3.2 Modifying the receptor. Adhesion can be inhibited by modifying the receptor in such a way that it no longer can specifically interact with the adhesin. Trypsin treatment of receptor containing intestinal material inhibited the interaction between the material and the K88 adhesin 131. This suggests that proteolytic activity may have a protective function against microbial colonisation of the small intestine 27. Proteolytic treatment with bromelain, has been shown to reduce the receptor activity for K88 fimbriae in vivo 27. It could therefore 41 function as a prophylactic treatment against E. coli K88 induced diarrhoea 132. Protease activity from Saccharomyces boulardii has also been suggested to remove or reduce brush border glycoproteins involved in adhesion of pathogens ot the mucosa, analogous to the inhibition of Clostridium difficile toxin A binding 146. Displacement of receptors has also been observed 133. Bovine casein derivatives displaced human serum albumin from saliva coated hydroxyapatite, thereby interfering with the adhesion of S. sobrinus and S. sanguis. The displacement can be explained in terms of the Vroman effect, which describes the displacement of adsorbed proteins by more surface active ones. 6.3.3 Specific blocking of the receptor. A receptor can be blocked specifically by addition of free adhesin. The adhesin will bind to the receptor, blocking it from any interaction with adhesin carrying pathogens. Outer membranes have been shown to be competitive inhibitors of E. coli adhesion to epithelial cells 174. Lipoteichoic and teichoic acid have been found to inhibit adhesion of Staphylococcus aureus to epithelial cells by binding to the staphylococcal receptor site 2. Lectins with specificity for different carbohydrates have been found to inhibit the adhesion of E. coli to brush-border membranes. Interestingly, the inhibition was independent of the carbohydrate specificity and solely depended upon the capacity of the lectin to bind to the brushborder 94. One could postulate that the lectin may not bind to the receptor, but inhibits the adhesion by steric hindrance. 6.3.4 Colonisation resistance. Non-specific blocking of the receptor. The mechanisms which together account for the phenomenon of colonisation resistance (as described in 4.2.1) will make it difficult for a new microorganism to establish. Interfering with the adhesion of the new coming microorganism can be done most effective by blocking its potential receptors, either specifically or non-specifically, i.e. steric hindrance. This concept has been extensively investigated for lactobacilli. It has been shown that lactobacilli and some other organisms [88] inhibit the adhesion of pathogens by blocking their receptors. Reid and co-workers investigated the effect of the normal microflora and lactobacilli on the adhesion of uropathogens to uroepithelial cells. They found that pre-incubation of uroepithelial cells with Lactobacillus sp. or a diphtheroid organism isolated from the normal microflora were able to inhibit the adhesion of Gram-negative uropathogens to uroepithelial cells in suspension 25. They also achieved complete or partial inhibition of 42 adhesion of Gram-negative uropathogens using bacterial cell wall fragments isolated from a Lactobacillus strain, although whole viable cells were more effective than cell wall fragments. It was concluded that lipoteichoic acid was responsible for the adhesion of Lactobacillus cells to uroepithelial cells, but that steric hindrance was the major mechanism of adhesion inhibition 26. In a later study, however, it was shown that steric hindrance is not likely to be the sole mechanism of action. Of equally adherent Lactobacillus strains, the larger strain, which should cover a larger surface area of the host cell, did not cause the largest inhibition of adhesion. Adhesiveness appears to be a major determinant in adhesion inhibition 151. Heat killed Lactobacillus acidophilus has been found to inhibit the adhesion of enteropathogens to tissue cultures. Fourniat and co-workers 61 found that the spent culture liquid, pH adjusted to 5, was necessary for the adhesion inhibitory effect. The liquid itself had no effect on the adhesion of enteropathogenic E. coli. Since lysed lactobacilli were not able to inhibit adhesion, it was concluded that the inhibition was not a specific competition for a common receptor but rather steric hindrance of binding sites for the E. coli. Servin and co-workers 10, 11, 29, 33, 34 found that heat killed and live L. acidophilus cells and bifidobacteria were able to inhibit the adhesion and invasion of enteropathogenic E. coli, S. typhimurium, Yersinia pseudotuberculosis and Listeria monocytogenes to tissue culture cells. They too concluded that blocking of the receptors by steric hindrance is the most likely mechanism. However, all experiments were done in the presence of culture liquid and no mention is made as to whether the pH was adjusted. It is also not clear what control was used in the experiments since it could have been treatment with fresh broth, buffer or may not have been treated. Activity was found to be dose-dependent, however dilution of the bacterial suspension with fresh broth is likely to change the pH of the suspension. It has been shown that pH is a very important factor in adhesion to tissue culture cells 77, 112. 6.3.5 Lactobacillus cell wall fragments. Non-specific blocking of the receptor. As mentioned above, whole lactobacilli, live and heat-killed are able to block adhesion of enteropathogens to tissue culture cells. Cell wall fragments have also been found to inhibit enteropathogen adhesion 26. Blomberg and co-workers 14 found a component in Lactobacillus spent culture liquid that inhibited the adhesion of K88 expressing E. coli cells to porcine ileal mucus. It was concluded to be a heat-stable protein with a molecular weight of more than 250K. The mechanism of action was proposed to be blocking of adhesion by steric hindrance through 43 interactions with mucus components. The phenomenon was studied in more detail as described in papers II and III. Lactobacilli were grown in a semi-defined medium 105, with a high acetate content, namely 15 g.l-1 sodium acetate. The activity of the spent culture liquid was tested in an in vitro adhesion assay as described in section 5.4. It appeared that acetate was essential for the presence of the component in the spent culture liquid. Acetate also caused a rapid decline in the number of viable cells in the culture. The K88 adhesion inhibitory component could also be isolated first at late log-phase, no stationary-phase was observed (Fig. 1 paper II). It can therefore be speculated that the active component originates from dead Lactobacillus cells. It is apparent that acetate which is added to Lactobacillus selective media to inhibit growth of other microbes also creates conditions which have negative effects on lactobacilli. The activity was found in fractions corresponding to a size of approximately 1700K when dialyzed and concentrated spent culture liquid from L. fermentum 104r was fractionated by gel filtration. When the concentrate was treated with pronase prior to fractionation, the size was reduced to approximately 1100K. This indicates that protein could be associated with the component but appears not to be necessary for activity. After pronase treatment, only small amounts of protein were found to be present in the fractions, but considerable amounts of carbohydrate. Previously the activity had been found to be heat stable (15 min. 80C) 14. In fact, the activity was found to be extremely heat stable with no loss of activity after 20 min. at 121C. The fractions were found to contain glucose, N-acetylglucosamine and galactose, carbohydrates typically found in Lactobacillus cell wall 111. Treatment with lysozyme and glucose oxidase, but not endo--galactosidase, removed the activity. These findings suggest that the active component is of carbohydrate nature, and very likely to be a cell wall component. Because cell wall fragments from homogenized cells were not active and because the activity could not be removed from the spent culture liquid by centrifugation, 27000 x g, 1.5h 14, it was concluded that the active component was soluble in nature. Of the cell wall components, it appeared not to be teichoic acid since the activity was found to be acid stable. Treatment with 0.05 M HCl at 60C for 20 min. hydrolyses teichoic acids, the adhesion inhibiting activity was however not affected by this treatment 138. The adhesion of E. coli K88 to immobilised mucus pretreated with Lactobacillus spent culture liquid was found to be 9.23 2.81 (n = 3) as compared to the medium treated control. Spent culture liquid treated with HCl reduced the adhesion to a similar level 5.97 1.85 (n = 3). The precise mode of action still remains to be explained. Using ellipsometry measurements, it was not possible to detect any change in 44 thickness of the immobilised mucus film upon treatment with spent culture liquid. Further more this treatment did not produce any change in hydrophobicity of the immobilised mucus film. When material in the spent culture liquid was radiolabelled, a small part of the activity was found to bind to the immobilised mucus. Preliminary results from Scatchard plot analysis, using radiolabelled spent culture liquid, suggest binding to a single receptor. Spent culture liquid appeared to inhibit the adhesion to certain ileal mucus fractions considerably more than to other fractions (Fig. 5 paper II). A preliminary conclusion is that the active component binds to certain mucus components and blocks the adhesion by steric hindrance. This hypothesis is further supported by the finding that spent culture liquid from L. fermentum 104r was able to inhibit adhesion mediated by all K88 fimbrial serotypes and mediated by CFA/II and SfaII fimbriae, the latter two being human pathogens (paper III). The adhesion of the target strains, CFA/I, CFA/II, CFA/IV and SfaII fimbriae, was inhibited by spent culture fluid from strain HBL8 more than by spent culture fluids from the other tested Lactobacillus strains . Spent culture liquids from all tested Lactobacillus strains were able to inhibit SfaII-fimbriae-mediated adhesion. (paper III). What argues against the steric hindrance theory is the finding that adhesion to mucus is also inhibited when mucus is treated with spent culture liquid as a suspension prior to immobilization. When mucus was first immobilised and then treated with, 5x concentrated, spent culture liquid, as in all previous cases, adhesion of E. coli K88 was 20.57% 2.76 (n = 3). When mucus was first incubated with, 5x concentrated, spent culture liquid, as a suspension, and subsequently immobilised, adhesion of E. coli K88 was found to be 24.68% 2.47 (n = 3). Consequently steric hindrance can only occur if the active component and E. coli bind to the same molecule but to different epitopes. Blocking of the K88 receptors during immobilization is not likely, since this was performed at 4C and no significant activity could be observed at 0C (paper II). It was also observed that many other lactobacilli of enteric origin were able to inhibit K88ac adhesion. Strains from porcine origin performed best, but even strains from human origin were able to block this adhesion. Host specificity appears not to be of major importance for this property. Strains from non-enteric origin were not able to cause any inhibiton of adhesion of E. coli K88ac (paper III). For in vivo application of this substance, in situ production of the substance would be preferable. This is most likely to occur for a strain originating from the same host since it is more likely to colonise and therefore grow in situ. Unfortunately, in vivo production of the inhibitory component may not occur since, after growth in porcine ileal mucus no inhibitory activity could be observed. This could be linked with the fact that high concentrations of acetate are necessary. 45 Another possibility for in vivo use is the direct oral administration of the substance. This should preferably be administered prophylactically, since already bound E. coli are not released. If used when the enteropathogen is already present, it may limit the proliferation of the pathogen. In an attempt to mimic the intestinal situation, porcine ileal mucus was immobilised on glass beads in a column. After washing, the column was filled with spent culture liquid and incubated at 37C for 1,5 h. Fractions were collected and tested for K88 adhesion inhibiting activity (Figure 6.1), the inhibitory activity was found to be significantly reduced. The fractions were concentrated and applied to a Sepharose CL 4B column. The carbohydrate pattern was found to be slightly changed (Figure 6.2). These findings indicate that the mucus is affecting the activity present in spent culture liquid. As mentioned previously, mucus contains lysozyme. The lysozyme may have reduced the activity. Furthermore, the active component may have bound to the immobilised mucus. Consequently, it may be concluded that large amounts of active component are probably necessary for in vivo activity in order to saturate the mucus and/or compensate for loss of activity due to lysozyme activity. Administration of the substance has the advantage that the best producing strain can be used, whereas if the live microbe is used one would need a strain which can colonise the host. 140 Adhesion (%) 120 100 80 60 40 20 0 spent culture liquid 2 4 Fraction (1ml) 6 Figure 6.1 Adhesion of Escherichia coli 1107 K88ac to mucus treated with Lactobacillus fermentum 104r spent culture liquid fractions from a column with immobilised mucus. 46 110 100 Carbohydrate ( g/ml) 90 80 70 60 50 40 30 20 10 0 -10 0 10 20 30 40 50 60 70 80 90 Fraction (2ml) Figure 6.2 Carbohydrate profile of Lactobacillus fermentum 104r spent culture liquid () and L. fermentum 104r spent culture liquid from a column with immobilised mucus (). Fractionation was performed using Sepharose CL 4b. 6.3.6 -lactoglobulin. Non-specific blocking of the receptor. As mentioned above, many milk components have been found to inhibit adhesion of a variety of enteropathogens, in most cases by blocking the adhesin of the bacterium. In this section the effect of milk components, in particular -lactoglobulin, on the host receptor will be discussed. Giampaglia and Silva 72 showed that pretreatment of HeLa cells with human colostrum inhibited the adhesion to these cells of all tested E. coli strains, by 37 to 90%, depending on the strain tested. Simultaneous incubation of E. coli and HeLa cells in the presence of human colostrum or milk, was found to be more effective, with the adhesion of E. coli inhibited by up to 97%. Oligosaccharides and sIgA were suggested to be the active components, albeit no attempt was made to identify them or the mechanism of inhibition. As mentioned in section 3.2.2a, the major function of -lactoglobulin (-lg) is proposed to be the binding of retinol. In paper IV and V it is shown that -lg also appears to have an antimicrobial activity. 47 Bovine colostrum was defatted by centrifugation and acidified to precipitate caseins. The pH of the resultant whey was re-adjusted to neutral, and the whey was fractionated by ultra filtration into fractions with different molecular weights: <100K, <30K and <10K. The fractions were tested for their ability to inhibit the adhesion of SfaII-fimbriated E. coli to human ileostomy glycoproteins. SfaII mediated adhesion was found to be inhibited by colostrum whey, its <100K and <30K fractions, but not by the <10K fraction. Adhesion was reduced to approximately 25% of the buffer treated control. After purification, using anion exchange chromatography, an 18K protein was found, with an isoelectric point of approximately 5.75. The amino acid sequence of the NH2 terminus, was found to be identical to -lg. In paper V, -lg was purified from milk whey by gel filtration. The activity was found to be very heat stable with no loss of activity after 40 min. at 120C. It was suggested that disulphide bridges may stabilise parts of the protein. It was hypothesized that these were important for the observed adhesion inhibitory activity. Boiling of the protein in the presence of a reducing agent, -mercaptoethanol, abolished the activity. Thus supporting the hypothesis. Limited digestion of -lg by CNBr at methionine and cysteine residues, also abolished activity, further supporting the hypothesis that the areas around the cysteine residues are important for the activity of -lg. Pre-treatment of E. coli SfaII did not inhibit adhesion, however, inhibition was noted after pre-treatment of immobilised ileostomy glycoproteins. -lg appears not to recognize N-acetyl-neuraminyl-2,3-lactose (NANA), the receptor of SfaII-fimbriae. Pre-incubation of -lg with NANA did not affect the inhibitory activity. -lg, however, does bind to the immobilised ileostomy glycoproteins, most likely by using multiple binding sites. This was concluded from a Scatchard plot (Fig. 2, paper V) and from binding studies to immobilised ileostomy glycoproteins fractions (Fig. 3, paper V). Commercially available -lg was found to also inhibit SfaII mediated adhesion, in a concentration dependent manner (Fig. 1 paper V). The optimal concentration was found to be 10-50 g.ml-1. Similar concentration dependent effects can be observed for antibody-antigen reactions 23, suggesting a fixed ratio between -lg and its target. Three out of four groups of ileostomy glycoprotein fractions that bound -lg also bound E. coli SfaII. Consequently, it was hypothesised that -lg recognizes a different epitope on the same molecule as E. coli SfaII, thus blocking the SfaII receptor by steric hindrance. General steric hindrance is unlikely since of all tested strains, only the adhesion of E. coli expressing SfaI or SfaII and S. enteritidis and S. typhimurium was inhibited, with the adhesion of the latter two being inhibited to a very limited extend. The hypothesis of 48 two epitopes on one molecule is strengthened by the observation that ileostomy glycoproteins do not inhibit the adhesion of E. coli SfaII to enterocytes when suspended with -lg (see below). Considering the fact that -lg is present in cow milk and not in human milk, it is surprising to find that the adhesion of the above mentioned human pathogens was inhibited, while the adhesion of the two tested bovine enteropathogens was not affected. Recently, 3-hydroxyphthalic anhydride modified -lg has been found to block the CD4 cell receptor for HIV 135, which is not a bovine pathogen. As previously mentioned, mucus is believed to block bacterial adhesins 65, 122, 150, 156. Mucins have been found to inhibit adhesion of SfaII fimbriae to buccal cells 169, 170, 171. It can therefore be hypothesised that by reducing the binding between mucus and the fimbrial adhesin, SfaII fimbriated E. coli can penetrate the mucus gel and bind to the underlying enterocytes. This would thus promote infection. To test this hypothesis, E. coli SfaII was allowed to bind to Caco-2 cells in the presence of HH-buffer, ileostomy glycoproteins (≡mucus) or the combination of ileostomy glycoproteins and -lg. Adhesion of E. coli in the presence of ileostomy glycoproteins reduced the adhesion to 56.14% 15.44 (n=7) as compared to the control experiment in the presence of HH-buffer. In the presence of both ileostomy glycoproteins and -lg, the adhesion was not significantly altered; 94.21 14.47 (n=7), compared to the control. These results confirm that mucus does block binding to the underlying enterocytes. They also suggest that the blocking function of intestinal mucus may be impaired by -lg. On the other hand, E. coli SfaII may not be able to make an initial binding to the mucus in the presence of -lg and thus be unable to colonise and penetrate the mucus layer to subsequently cause infection. The in vivo significance of -lg as an adhesion inhibitor remains, however, to be assessed. -lg might be used prophylactically, or therapeutically in combination with antibiotics to avoid further translocation.The latter would be important if risk for antibiotic resistance exists. At present, -lg is considered to be one of the causes of cow milk allergy 63. Also, if not only the intestine but also the oral cavity is the site of infection 80, it should be tested whether -lg even inhibits the adhesion to buccal cells, in order to be effective. 49 7 Conclusions The thesis focuses on substances that inhibit the initial binding of pathogenic E. coli to intestinal mucus in vitro. Since adhesion is considered to be a prerequisite for pathogenesis 9, 59, 93, 152, interfering with the adhesion of a pathogen may prevent the establishment of disease in an early stage. Many substances have been reported to inhibit the adhesion of pathogens in vitro. Most substances which are described to have an adhesion inhibitory effect against enteropathogens, work as receptor analogues and block the bacterial adhesin (section 6.2.1). The two substances described in this thesis, appear to block the receptor(s) sites of the intestinal mucus. It was proposed that the substances block the receptor(s) for the pathogen by steric hindrance. Identification of the receptors for the adhesion inhibitory substances would provide further information on the mechanism involved. It was concluded that spent culture liquid from L. fermentum 104r contains polysaccharides with an estimated Mr of 1700 K that mediate the adhesion inhibitory activity. The polysaccharides are likely to be soluble cell wall fragments, since the activity is affected by lysozyme and they contain monosaccharides typically found in cell walls (paper II). The adhesion inhibitory activity was mainly directed against the different K88 fimbrial serotypes and SfaII fimbriae. Other Lactobacillus strains of intestinal origin were also found to produce similar activity (paper III). L. fermentum 104r was also found to produce a high molecular weight substance that potentiates the bactericidal activity of the organic acids produced by its metabolism. The in vivo significance of this effect remains to be investigated with unanswered questions as to whether the substance is produced in vivo and whether the in vivo concentration of organic acids are sufficiently high to exert any growth inhibiting effect (paper I). The substance mediating the adhesion inhibitory activity in bovine colostrum, was identified as -lactoglobulin (-lg), a major cow milk whey protein (paper IV). -lg was found to bind to several intestinal mucus proteins, and to inhibit the adhesion of SfaII and, to a lesser extent, SfaI fimbriae (paper V). The two adhesion inhibitory substances described in this thesis, have not been tested in vivo. Preliminary data indicate potential problems with in vivo use. The Lactobacillus cell wall fragments are sensitive to lysozyme (paper II). Lysozyme present in the intestine might inactivate the fragments before they reach the site where they should exert their activity; the ileum. Chemical modification that would protect against hydrolysis by lysozyme, may solve this problem. -lg may be used prophylactically. -lg is, 50 however, considered to be a cause of allergy to cow milk 63, and may therefore not be desirable to be used prophylactically. A fundamental question to be answered is if reduced adhesion to mucus is beneficial for the host. The ability of bacteria to bind to mucus constituents might be advantageous for the host as it prolongs the time of mucus penetration and may prevent pathogens from binding to the underlying epithelium. For -lg it has been shown (see section 6.3.6) that reduced binding to intestinal mucus may actually enhance the binding abilities of SfaII fimbriae expressing E. coli to the underlying epithelial cells. On the other hand, the binding may also be advantageous for the bacterium since it facilitates colonisation of the mucus layer. In order for such colonisation to take place, the bacterium must either multiply at a rate exceeding the rate at which mucus is sloughed into the lumen of the intestine or actively penetrate the mucus 65, 122, 150. The balance between these two effects may be different for different bacterial strains. In summary, it can be concluded that L. fermentum 104r spent culture liquid contains substances that potentiate the bactericidal activity of organic acids and contains polysaccharides that inhibit K88-fimbriae-mediated adhesion. Furthermore, it was also demonstrated that -lg present in bovine colostrum and milk, inhibits SfaII-fimbriae-mediated adhesion. 51 8 Acknowledgements I am very grateful to my supervisor Patricia ‘Trish’ Conway, who replied positive when I was looking for a place to do the practical part of my undergraduate studies and invited me to continue as ‘doktorand’. I also admire her indestructible optimism. Also my sincere gratitude to Seppo Salminen for making my move to Finland possible, being co-supervisor during the last two years and for help with practical things like baby clothes and a pram. Thanks also to Lennart Adler and Staffan Kjelleberg who were always willing to help with lots of things during my Ph.D. studies. Thanks to all present and previous members of ‘mag-tarm gruppen’ () for the pleasant atmosphere to work in and all the small talk: Allan, AnnCathrin, Anna, Annika, Camilla, Christer, David, Elisabeth, Lars, Maurilia, Paul, Ruth, and especially to Agneta; for your help with lots of things even when I was in Finland, and Lena and Anders; for all the valuable things you have told and learned me concerning intestinal microbiology, when I was still a novice. Thanks also to the members of ‘bacterial virulence’; Elise, Jarmo, Maria, Pia, Reija, Saija and Yasmin and of course Miikki for the discussions and your good sense of humor. And to the people at Viable Ltd., in particular Ari; for the non-sense discussions we’ve had, and Elina; for introducing me into the mysterious world of cell culture and for being a pleasant colleague and friend. I also wish to thank all the people who helped me with the practical and bureaucratic things involved in moving abroad; Anita, Maureen, Lise and Inga in Göteborg and Kaija in Åbo/Turku. But most of all for their good spirit, which positively affects the respective departments. 52 9 References 1 p. 2 3 4 5 6 7 8 9 10 11 Allen, A. 1984 The structure and function of gastrointestinal mucus. 3-11 In: E.C. Boedeker (ed.) Attachment of organisms to the gut mucosa. Vol. II. CRC Press. Boca Raton. Aly, R., H.R. Shinefield, C. Litz and H.I. Maibach. 1980 Role of teichoic acid in the binding of Staphylococcus aureus to nasal epithelial cells. J. Infect. Dis. 141:463-465. Anderson, M.J., J.S. Whitehead and Y.S. Kim. 1980 Interaction of Escherichia coli K88 antigen with porcine intestinal brush border membranes. Infect. Immun. 29:897-901. Ashkenazi, S. and D. Mirelman. 1987 Nonimmunologlobulin fraction of human milk inhibits the adherence of certain enterotoxigenic Escherichia coli strains to guinea pig intestinal tract. Pediatr. Res. 22:130-134. Avorn, J. 1996 The effect of cranberry juice on the presence of bacteria and white blood cells in the urine of elderly women: what is the role of bacterial adhesion? Proc. Bat-Sheva seminar: Toward antiadhesion therapy of microbial diseases. p. 32-33. Zichron Yaakov, Israel. Axelsson, L.T. 1993 Lactic acid bacteria: classification and physiology. p. 1-63. In: S. Salminen and A. von Wright (eds.) Lactic acid bacteria. Marcel Dekker Inc. New York. Barrow, P.A., B.E. Brooker, R. Fuller and M.J. Newport. 1980 The attachment of bacteria to the gastric epithelium of the pig and its importance in the microecology of the intestine. J.Appl. Bact. 48:147-154. Bäumler, A.J., R.M. Tsolis and F. Hefron. 1996 Contribution of fimbrial operons to attachment to and invasion of epithelial cell lines by Salmonella typhimurium. Infect. Immun. 64:1862-1865. Beachey, E.H. 1981 Bacterial adherence: Adhesin-receptor interactions mediating the attachment of bacteria to mucosal surfaces. J. Infect. Dis. 143:325-345. Bernet, M.-F., D. Brassart, J.-R. Neeser and A.L. Servin. 1993 Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibiton of enteropathogen-cell interactions. Appl. Env. Microbiol. 59:4121-4128. Bernet, M.-F., D. Brassart, J.-R. Neeser and A.L. Servin. 1994 Lactobacillus acidophilus LA 1 binds to cultured human intestinal cell lines and inhibits cell attachment and cell invasion by enterovirulent bacteria. Gut. 35:483-489. 53 12 13 14 15 16 17 18 19 20 21 22 23 54 Bijlsma, I.G.W., A. de Nijs, C. van der Meer and J.F. Frik. 1982 Different pig phenotypes affect adherence of Escherichia coli to jejunal brush borders by K88ab, K88ac or K88ad antigen. Infect. Immun. 37:891-894. Blom, H. and C. Mörtvedt. 1991 Anti-microbial substances produced by food associated micro-organisms. Biochemical society Transactions 19:694-698. Blomberg, L., A. Henriksson and P.L. Conway. 1993 Inhibition of adhesion of Escherichia coli K88 to piglet ileal mucus by Lactobacillus spp. Appl. Env. Microbiol. 59:34-39. Blomberg, L., H.C. Krivan, P.S. Cohen and P.L. Conway. 1993 Piglet ileal mucus contains protein and glycolipid (galactosylceramide) receptors specific for Escherichia coli K88 fimbriae. Infect. Immun. 61:2526-2531. Breines, D.M. and J.C. Burnham. 1994 Modulation of Escherichia coli type 1 fimbrial expression and adherence to uroepethelial cells following exposure of logarithmic phase cells to quinolones at subinhibitory concentrations. J. Antimicrobial Chemother. 34:205221. Buchanan, T.M. 1978 Antigen specific serotyping of Neisseria gonorrhoea. Use of an enzyme-linked immunosorbent assay to quantitate pilus antigens on gonococci. J. Infect. Dis. 138:319-325. Busscher, H.J. and A.H. Weerkamp. 1987 Specific and non-specific interactions in bacterial adhesion to solid substrata. FEMS Micrbiol. Rev. 46:165-173. Caloca, M.J., J. Soler and S. Suárez. 1996 Adhesion of K88ab to guinea pig erythrocytes: Effect on membrane enzyme activities. Infect. Immun. 64:3416-4318. Carlsson, I., Y. Iwami and T. Yamada. 1983 Hydrogen peroxide excretion by oral streptococci and effect of lactoperoxidasethiocyanate- hydrogen peroxide. Infect. Immun. 40:70-80. Carlstedt, I., S. Elmquist, I. Ljusegren and G.C. Hansson. 1991 Structure and properties of rat gastrointestinal mucins. p. 23-28. In: T. Wadström, P.H. Mäkelä, A.-M. Svennerholm and H. Wolf-Watz (eds.) Molecular pathogenesis of gastrointestinal infections. Plenum Press. London. Carlstedt-Duke, B. 1989 The normal microflora and mucin. p. 109-127. In: R. Grubb, T. Midtvedt and E. Norin (eds.) The regulatory and protective role of the normal microflora. M. Stockton Press, New York. Carpenter, P.L. 1965 Immunology and serology. W.B. Saunders Co, London. 24 25 26 27 28 29 30 31 32 33 34 Carsenti-Etesse, H., J. Durant, E. Bernard, V. Mondain, J. Entenza and P. Dellamonica. 1992 Effect of subinhibitory concentrations of cefamandole and cefuroxime on adherence of Staphylococcus aureus and Staphylococcus epidermidis to polystyrene culture plates. Eur. J. Clin. Microbiol. Infect. Dis. 11:732-737. Chan, R.C.Y., A.W. Bruce and G. Reid. 1984 Adherence of cervical, vaginal and distal urethral normal microbial flora to human uroepithelial cells and the inhibition of adherence of Gram-negative uropathogens by competitive exclusion. J. Urol. 131:596-601. Chan, R.C.Y., G. Reid, R.T. Irvin, A.W. Bruce and J.W. Costerton. 1985 Competitive exclusion of uropathogens from human uroepithelial cells by Lactobacillus whole cells and cell wall fragments. Infect. Immun. 47:84-89. Chandler, D.S., T.L. Mynott, R.K.J. Luke and J.A. Craven. 1994 The distribution and stability of Escherichia coli K88 receptor in the gastrointestinal tract of the pig. Vet. Microbiol. 38:203-215. Chang, R. 1981 Physical chemistry with applications to biological systems. Macmillan Publishing Co., Inc. New York. Chauvière, G., M.-H. Coconnier, S. Kerneis, A. DarfeuilleMichaud, B. Joly and A.L. Servin. 1992 Competitive exclusion of diarrhoeagenic Escherichia coli (ETEC) from human enterocyte-like Caco-2 by heat-killed Lactobacillus. FEMS Microbiol. Lett. 91:213-218. Chauvière, G., M.-H. Coconier, S. Kernéis, J. Fourniat and A.L. Servin. 1992 Adhesion of human Lactobacillus acidophilus strain LB to human enterocyte-like Caco-2 cells. J. Gen Microbiol. 138:1689-1696. Cherrington, C.A., V. Allen and M. Hinton. 1992 The influence of temperature and organic matter on the bactericidal activity of shortchain organic acids on salmonellas. J. Appl. Bact. 72:500-503. Clegg, S., T.K. Korhonen, D.B. Hornick and A.-M. Tarkkanen. 1994 Type 3 fimbriae of the Enterobacteriaceae. p 97-103. In: P. Klemm (ed.) Fimbriae: adhesion, genetics, biogenisis and vaccines. CRC Press. Boca Raton. Coconnier, M.-H., M.-F. Bernet, G. Chauvière and A.L. Servin. 1993 Adhering heat-killed human Lactobacillus acidophilus, strain LB, inhibits the process of pathogenicity of diarrhoeagenic bacteria in cultured human intestinal cells. J. Diarrhoeal Dis. Res. 11:235-242. Coconnier, M.-H., M.-F. Bernet, S. Kernéis, G. Chauvière, J. Fourniat and A.L. Servin. 1993 Inhibition of adhesion of enteroinvasive pathogens to human intestinal Caco-2 cells by Lactobacillus acidophilus strain LB decreases bacterial invasion. FEMS Microbiol. Lett. 110:299-306. 55 35 36 37 38 39 40 41 42 43 44 45 46 47 56 Cohen, P.S. and D.C. Laux. 1995 Bacterial adhesion to and penetration of intestinal mucus in vitro. Methods Enzymol. 253:309-314. Collins, F.M. and P.B. Carter. 1978 Growth of salmonellae in orally infected germfree mice. Infect. Immun. 21:41-47. Collinson, S.K., P.C. Doig, J.L. Doran, S. Clouthier, T.J. Trust and W.W. Kay. 1993 Thin, aggregative fimbriae mediate binding of Salmonella enteritidis to fibronectin. J. Bact. 175:12-18. Conway, P.L. 1994 Function and regulation of the gastrointestinal microbiota of the pig. Proc. VIth International symposium on digestive physiology in pigs. p. 231-240. Bad Doberan, Germany. Conway, P.L. and R.F. Adams. 1989 Role of erythrosine in the inhibition of Lactobacillus fermentum strain 737 to mouse stomach tissue. J. Gen. Bact. 135:1167-1173. Conway, P.L. and A. Henriksson. 1994 Strategies for the isolation and characterisation of functional probiotics. p.75-93. In: S.A.W. Gibson (ed.) Human health: The contribution of microorganisms. Springer-Verlag. London. Conte, M.P., C. Longhi, V. Buonfiglio, M. Polidoro, L. Seganti and P. Valenti. 1994 The effect of iron on the invasiveness of Escherichia coli carrying the inv gene of Yersinia pseudotuberculosis. J. Med Microbiol. 40:236-240. Coppa, G.V., O. Gabrielli, P. Giorgi, C. Catassi, M.P. Montanari, P.E. Varaldo and B.L. Nichols. 1990 Preliminary study of breastfeeding and bacterial adhesion to uroepithelial cells. Lancet. 335:569-571. Craven, S.E., N.A. Cox, J.S. Bailey and L.C. Blankenship. 1992 Binding of Salmonella strains to immobilized intestinal mucosal preparations from broiler chickens. Avian Dis. 36:296-303. Cravioto, A., A. Tello, H. Villafán, J. Ruiz, S. del Vedovo and J.-R. Neeser. 1991 Inhibition of localized adhesion of enteropathogenic Escherichia coli to Hep-2 cells by immunoglobulin and oligosaccharide fractions of human colostrum and breast milk. J. Infect. Dis. 163:1247-1255. Creeth, J.M. 1978 Constituents of mucus and their separation. Br. Med. Bull. 34:17-24. Cunningham, A.S., D.B. Jelliffe and E.F.P. Jelliffe. 1991 Breastfeeding and health in the 1980s: A global epidemiologic review. J. Pediatr. 118:659-666. Darfeuille-Michaud, A., D. Aubel, G. Chauvière, C. Rich, M. Bourgges, A. Servin and B. Joly. 1990 Adhesion of enterotoxigenic Escherichia coli to the human colon carcinoma cell line Caco-2 in culture. Infect. Immun. 58:893-902. 48 49 50 51 52 53 54 55 56 57 58 59 60 61 Dean, E.A. and R.E. Isaacson. 1985 Purification and characterization of a receptor for the 987P pilus of Escherichia coli. Infect. Immun. 47:98-105. de Graaf, F.K. and W. Gaastra. 1994 Fimbriae of enterotoxigenic Escherichia coli. p. 53-83. In: P. Klemm (ed.) Fimbriae: adhesion, genetics, biogenisis and vaccines. CRC Press. Boca Raton. Derjaguin, B.V. and L. Landau. 1941 Theory of the stability of strongly charged lyophobic soils and of adhesion of strongly charged particles in solutions of electrolytes. Acta Physic. Chemic. U.S.S.R. 14:633-662. Desnottes, J.F., D. LeRoy and N. Diallo. 1988 Effect of sub-minimal inhibitory concentrations of pefloxacin on the piliation and adherence of E. coli. Drugs Exp. Clin. Res. 14:629-634. de Wit, J.N. 1989 Functional properties of whey proteins. p.285-321. In: P.F. Fox (ed.) Developments in dairy chemistry-4; functional milk proteins. Elsevier Applied Science. London. Donnenberg, M.S. and J.B. Kaper. 1992 Enteropathogenic Escherichia coli. Infect. Immun. 60:3953-3961. Duguid, J.P., I.W. Smith, G. Dempster and P.N. Edmunds. 1955 Non-flagellar filamentous appendages (“fimbriae”) and haemagglutinating activity in Bacterium coli. J. Pathol. Bacteriol. 70:335-347. Duguid, J.P. and D.C. Old. 1994 Introduction: a historical perspective. p. 6-7. In: P. Klemm (ed.) Fimbriae: adhesion, genetics, biogenesis and vaccines. CRC Press. Boca Raton. Eisenhauer, P.B., S.S.S.L. Harwig and R.I. Lehrer. 1992 Cryptdins: antimicrobial defensins of the murine small intestine. Infect. Immun. 60:3556-3565. Eisenstein, B.I., I. Ofek and E.H. Beachey. 1981 Loss of lectin-like activity in aberrant type 1 fimbriae of Escherichia coli. Infect Immun. 31:792-797. Erickson, A.K., D.R. Baker, B.T. Bosworth, T.A. Casey, D.A. Benfield and D.H. Francis. 1994 Characterization of porcine intestinal receptors for the K88ac fimbrial adhesin of Escherichia coli as mucin-type sialoglycoproteins. Infect. Immun. 62:5404-5410. Finlay, B.B. and S. Falkow. 1989 Common themes in microbial pathogenicity. Microbiol. Rev. 53:210-230. Forstner, G., P. Sherman and J. Forstner. 1984 Mucus: function and structure. p. 13-21. In: E.C. Boedeker (ed.) Attachment of organisms to the gut mucosa. Vol. II. CRC Press. Boca Raton. Fourniat, J., C. Colomban, C. Linxe and D. Karam. 1992 Heat-killed Lactobacillus acidophilus inhibits adhesion of Escherichia coli to HeLa cells. Ann. Rech. Vet. 23:361-370. 57 62 63 64 65 66 67 68 69 70 71 72 73 74 75 58 Fox, P.F. 1989 The milk protein system. p.1-53. In: P.F. Fox (ed.) Developments in dairy chemistry-4; functional milk proteins. Elsevier Applied Science. London. Freed, D.J.L. 1984 Health hazards of milk. Baillière Tindall. London. Freedman, R.M., D.L. Ingram, I. Gross, R.A. Ehrenkranz, J.B. Warshaw and R.S. Baltimore. 1981 A half century of neonatal sepsis at Yale. Am. J. Dis. Child. 135:140-144. Freter, R. 1984 Factors involved in the penetration of the mucous layer inexperimental cholera. p. 43-50. In: E.C. Boedeker (ed.) Attachment of organisms to the gut mucosa. Vol. II. CRC Press. Boca Raton. Fuller, R. 1989 Probiotics in man and animals. J. Appl. Bacteriol. 66:365-378. Fuller, R. 1992 History and development of probiotics. P. 1-8. In: R. Fuller. (ed.) Probiotics: the scientific basis. Chapman and Hall, London. Fuller, R. P.A. Barrow and B.E. Brooker. 1978 Bacteria associated with the gastric epithelium of neonatal pigs. Appl. Env. Microbiol. 35:582-591. Gariépy, J., A.K. Judd and G.K. Schoolnik. 1987 Importance of disulfide bridges in the structure and activity of Escherichia coli enterotoxin ST1b. Proc. Natl. Acad. Sci. USA 84:8907-8911. Gaya, P., M. Medina and M. Nuñez. 1991 Effect of the lactoperoxidase system on Listeria monocytogens behaviour in raw milk at refrigeration temperatures. Appl. Env. Microbiol. 57:3355-3360. Gedek, B. 1986 Probiotika in der tiernährung; wirkung auf leisting und tiergesundheit. Kraftfutter 3:80-86. Giampaglia, C.M.S. and M.L.L. Silva. 1992 Effect of colostrum and human milk on the adherence to HeLa cells of common and rare enteropathogenic Escherichia coli serotypes found in Brazil. Rev. Microbiol., São Paulo. 23:211-216. Giannasca, K.T., P.J. Giannasca and M.R. Neutra. 1996 Adherence of Salmonella typhimurium to Caco-2 Cells: identification of a glycoconjugate receptor. Infect. Immun. 64:135-145. Giugliano, L.G., S.T.G. Ribeiro, M.H. Vainstein and C.J. Ulhoa. 1995 Free secretory component and lactoferrin of human milk inhibit the adhesion of enterotoxigenic Escherichia coli. J. Med. Microbiol. 42:3-9. Goodwin, A.E. and B.U. Pauli. 1995 A now adhesion assay using buoyancy to remove non-adherent cells. J. Immunol.Methods 187:213-219. 76 77 78 79 80 81 82 83 84 85 86 87 Gray, G.M. 1984 Oligosaccharidases of the intestinal membrane. p.111-119. In: E.C. Boedeker (ed.) Attachment of organisms to the gut mucosa. Vol. II. CRC Press. Boca Raton. Green, J.D. and T.R. Klaenhammer. 1994 Factors involved in adherence of lactobacilli to human Caco-2 cells. Appl. Env. Microbiol. 60:4487-4494. Greenberger, N.J. 1989 Gastrointestinal disorders; A pathophysiological approach. Medical Publishers. Chicago. Groves, M.L. and R. Greenberg. 1982 Complete amino acid sequence of bovine 2-microglobulin. J. Biol. Chem. 257:2619-2626. Guerina, N.G., T.W. Kessler, V.J. Guerina, M.R. Neutra, H.W. Clegg, S. Langermann, F.A. Scannapieco and D.A. Goldman. 1993 The role of pili and capsule in the pathogenesis of the neonatal infection with Escherichia coli K1. J. Infect. Dis. 148:395-405. Hacker, J., H. Kestler, H. Hoschützky, K. Jann, F. Lottspeich and T.K. Korhonen. 1993 Cloning and characterization of the S fimbrial adhesin II complex of an Escherichia coli O18:K1 meningitis isolate. Infect. Immun. 61:544-550. Hacker, J. and J. Morschäuser. 1994 S and F1C fimbriae. p. 27-36. In: P. Klemm (ed.) Fimbriae: adhesion, genetics, biogenesis, and vaccines. CRC Press. Boca Raton. Hanson, L.Å., I. Adlerberth, B. Carlsson, U. Dahlgren, M. HahnZoric, F. Jalil, S.R. Khan, P. Larsson, T. Midtvedt, D. Roberton, C. Svanborg-Edén and A. Wold. 1989 Colonization with Enterobacteriaceae and immune response, especially in the neonate. p. 59-69. In: R. Grubb, T. Midtvedt and E. Norin (eds.) The regulatory and protective role of the normal microflora. M. Stockton Press, New York. Havenaar, R., B. ten Brink and J.H.J. Huis in‘t Veld. 1992 Selection of strains for probiotic use. p.209-224. In: R. Fuller. (ed.) Probiotics: the scientific basis. Chapman and Hall, London. Hentges, D.J. 1992 Gut flora and disease resistance p. 87-108. In: R. Fuller. (ed.) Probiotics: the scientific basis. Chapman and Hall, London. Holmgren, J., A.-M. Svennerholm and C. Åhrén. 1981 Nonimmunoglobulin fraction of human milk inhibits bacterial adhesion (hemagglutination) and enterotoxin binding of Escherichia coli and Vibrio cholera. Infect. Immun. 33:136-141. Houwink, A.L. and W. van Iterson. 1950 Electron microscopical observations on bacterial cytology. II A study on flagellation. Biochem. Biophys. Acta 5:10-44. 59 88 Imundo, L., J. Barasch, A. Prince and Q. Al-Awqati. 1995 Cystic fibrosis epithelial cells have a receptor for pathogenic bacteria on their apical surface. Proc. Natl. Acad. Sci. USA. 92:3019-3023. 89 Isberg, R.R. 1994 Intracellular trafficking of Legionella pneumophila within Phagocytic cells. p. 263-278. In: V.L. Miller, J.B. Kaper, D.A. Portnoy and R.R. Isberg (eds.) Molecular genetics of bacterial pathogenesis. American Society for Microbiology, Washington DC. 90 Jacobsson, I., T. Lindberg, B. Benediktsson and B.G. Hansson. 1985 Dietary bovine -lactoglobulin is transferred to human milk. Acta Paediatr. Scan. 74:342-345. 91 Jack, R.W., J.R. Tagg and B. Ray. 1995 Bacteriocins of Grampositive bacteria. Microbiol Rev. 59:171-200. 92 Jensen, O.M. and A. Pærregaard. 1991 Inhibition of plasmidencoded adhesion of Yersinia enterocolitica by non-immunoglobulin fraction of human milk. APMIS 99:657-660. 93 Johnson, J.R. 1991 Virulence factors in Escherichia coli urinary tract infection. Clin. Microbiol. Rev. 4:80-128. 94 Jones, G.W. 1977 The attachment of bacteria to the surface of animal cells. p. 139-176. In: J.L. Reissig. (ed.) Microbial interactions; receptors and recognition, series B, Vol. 3, Chapman and Hall, London. 95 Jones, G.W. and R.E. Isaacson. 1983 Proteinaceous bacterial adhesins and their receptors. CRC Crit. Rev. Microbiol. 182:317-329. 96 Jones, C.H., F. Jacob-Dubuisson, K. Dodson, M. Kuehn, L. Slonim, R. Striker and S.J. Hultgren. 1992 Adhesin presentation in bacteria requires molecular chaperones and ushers. Infect. Immun. 60:4445-4451. 97 Jones, G.W. and J.M. Rutter. 1974 Contribution of the K88 antigen of Escherichia coli to enteropathogenicity; protection against disease by neutralizing the adhesive properties of K88 antigen. Am. J. Clin. Nutr. 27:1441-1449. 98 Jonsson, E. and P.L. Conway. 1992 Probiotics for pigs. p.259-316. In: R. Fuller. (ed.) Probiotics: the scientific basis. Chapman and Hall, London. 99 Järvinen, A.-K. and M. Sandholm. 1980 Urinary oligosaccharides inhibit adhesion of E. coli onto canine urinary tract epithelium. Invest. Urol. 17:443-445. 100 Kamau, D.N, S. Doores and K. M. Pruitt. 1990 Enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus by the lactoperoxidase system. Appl. Env. Microbiol. 56:2711-2716. 101 Klaenhammer, T.R. 1993 Genetics of bactericins produced by lactic acid bacteria. FEMS Microbiol. Rev. 12:39-86. 60 102 Klemm, P. and K.A. Krogfelt. 1994 Type 1 fimbriae of Escherichia coli. p. 9-26. In: P. Klemm (ed.) Fimbriae: adhesion, genetics, biogenesis, and vaccines. CRC Press. Boca Raton. 103 Korhonen, T.K., V. Väisänen-Rhen, M. Rhen, A. Pere, J. Parkkinen and J. Finne. 1984 Escherichia coli fimbriae recognizing sialyl galactosides. Infect. Immun. 159:762-766. 104 Korhonen, T.K., M.V. Valtonen, J. Parkkinen, V. Vaisänen-Rhen, J. Finne, F. Örskov, I. Örskov, S.B. Svenson and P.H. Mäkelä. 1985 Serotypes, hemolysin production, and receptor recognition of Escherichia coli strains associated with neonatal sepsis and menigitis. Infect. Immun. 48:486-491. 105 Kotarski, S.F. and D.C. Savage. 1979 Models for study of the specificity by which indigenous lactobacilli adhere to murine gastric epithelia. Infect. Immun. 26:966-975. 106 Kuehen, M.J., D. Haslam, S. Normark, and S.J. Hultgren. 1994 Structure, function and biogenesis of Escherichia coli P Pili. p. 37-51. In: P. Klemm (ed.) Fimbriae: adhesion, genetics, biogenesis, and vaccines. CRC Press. Boca Raton. 107 Kunz, C. and S. Rudloff. 1993 Biological functions of oligosaccharides in human milk. Acta Paediatr. 82:903-912. 108 Laux, D.C., E.F. McSweegan and P.S. Cohen. 1984 Adhesion of enterotoxigenic Escherichia coli to immobilized intestinal mucosal preparations: a model for adhesion to mucosal surface components. J. Microbiol. Meth. 2:27-39. 109 Laux, D.C., E.F. McSweegan, T.J. Williams, E.A. Wadolkowski and P.S. Cohen. 1986 Identification and characterization of mouse small intestine mucosal receptors for Escherichia coli K-12(K88ab). Infect. Immun. 52:18-25. 110 Lee, A. and S. Hazell. 1993 Pathogenicity of Helicobacter pylori: a perspective. Infect. Immun. 61:1601-1610. 111 Lehman, T.J.A., J.B. Allen, P.H. Plotz and R.L. Wilder. 1983 Polyarthritis in rats following the systemic injection of Lactobacillus casei cell walls in aqueous suspension. Arthritis Rheum. 26:12591265. 112 Lehto, E.M. and S.J. Salminen. 1995 The effect of Lactobacillus GG on the adhesion of Salmonella typhimurium to Caco-2 cell cultures. Proc. World congress on anaerobic bacteria and infections. p.113. San Juan, Puerto Rico. 113 Ligtenberg, M.J.L., H.L. Vos, A.M.C. Gennissen and J. Hilkens. 1990 Episialin, a carcinoma-associated mucin, is generated by a polymorphic gene encoding splice variants with alternative amino termini. J. Biol. Chem. 265:5573-5578. 61 114 Lindahl, M. and T. Wadström. 1985 Proc. VIII Symp. Glycoconugates, p. 366. Houston, Texas. 115 Lobley, R.W. 1991 The enterocyte and its brush border. In: T. Wadström, P.H. Mäkelä, A.-M. Svennerholm and H. Wolf-Watz (eds.) Molecular pathogenesis of gastrointestinal infections. Plenum Press. London. 116 Longhi, C., M.P. Conte, l. Seganti M. Polidoro, A. Alfsen and P. Valenti. 1993 Influence of lactoferrin on the entery process of Escherichia coli HB101 (pRI203) in HeLa cells. Med. Microbiol. Immunol. 182:25-35. 117Longhi, C., M.P. Conte, W. Bellamy, l. Seganti and P. Valenti. 1994 Effect of lactoferricin B, a pepsin-generated peptide of bovine lactoferrin, on Escherichia coli HB101 (pRI203) entry into HeLa cells. Med. Microbiol. Immunol. 183:77-85. 118 Loubeyre, C., J.F. Desnottes and N. Moureau. 1993 Influence of sub-inhibitory concentrations of antibacterials on the surface properties and adhesion of Escherichia coli. J. Antimicrob. Chemother. 31:37-45. 119 Macfarlane, G.T. and G.R. Gibson. 1993 Metabolic activities of the normal colonic flora. p. 17-52. In: S.A.W. Gibson. Human health: The contribution of microorganisms. Springer-Verlag. London. 120 Mack, D.R. and P.L. Blain-Nelson. 1995 Disparate in vitro inhibition of adhesion of enteropathogenic Escherichia coli RDEC-1 by mucins isolated from various regions of the intestinal tract. Pediatr. Res. 37:75-80. 121 Macy, J.M., I. Yu, C. Caldwell and R.E. Hungate. 1978 Reliable sampling methods for analysis of the ecology of the human alimentary tract. Appl. Env. Microbiol. 35:113-120. 122 Magnusson, K.-E. and I. Stjernström. 1982 Mucosal barrier mechanisms. Interplay between secretory (SIgA), IgG and mucins on the surface properties and association of salmonellae with intestine and granulocytes. Immunology 45:239-248. 123 Malmsten, M., E. Blomberg, P. Claesson, I. Carlstedt and I. Ljusegren. 1992 Mucin layers on hydrophobic surfaces studied with ellipsometry and surface force measurements. J. Coll. Interface Sci. 151:579-590. 124 Mantle, M., D. Mantle and A. Allen. 1981 Polymeric structure of pig small-intestine mucus glycoprotein. Biochem. J. 195:277-285 125 Mantle, M. and S.D. Husar. 1994 Binding of Yersinia enterocolitica to purified, native small intestinal mucins from rabbits and humans involves interactions with mucin carbohydrate moiety. Infect. Immun. 62:1219-1227. 62 126 Mantle, M. 1996 The anti-adherent role of intestinal mucus: mechanisms and physiopathology. Mucus Dialogue On-line 2:1-6. 127 Martin, G.P., C. Marriott and I.W. Kellaway. 1978 Direct effect of bile salts and phospholipids on the physical properties of mucus. Gut 19:103-107. 128 Morr, C.V. 1989 Whey proteins: manufacture. p. 245-284. In: P.F. Fox (ed.) Developments in dairy chemistry-4; functional milk proteins. Elsevier Applied Science. London. 129 Mukkur, T.K.S., D.L. Watson, K.S. Saini and A. K. Lascelles. 1985 Purification and characterization of goblet-cell mucin of high Mr from the small intestine of sheep. Biochem. J. 229:419-428. 130 Mullvihill, D.M. 1989 Caseins and caseinates: manufacture. p. 97130. In: P.F. Fox (ed.) Developments in dairy chemistry-4; functional milk proteins. Elsevier Applied Science. London. 131 Mynott, T.L. 1992 Protease and prevention of diarrhoea caused by enterotoxigenic Escherichia coli. PhD Thesis, La trobe University, Bundoora, Australia. 132 Mynott, T.L., R.K.J. Luke and D.S. Chandler. 1996 Oral administration of protease inhibits enterotoxigenic Escherichia coli receptor activity in piglet small intestine. Gut 38:28-32. 133 Neeser, J.-R., M. Golliard, A. Woltz, M. Rouvet, M.-L. Dillmann and B. Guggenheim. 1994 In vitro modulation of oral bacterial adhesion to saliva-coated hydroxyapatite beads by milk casein derivatives. Oral Microbiol. Immunol. 9:193-201. 134 Nielsen, E.M., J. Schlundt, A. Gunvig and B.L. Jacobsen. 1994 Epithelial, mucus and lumen subpopulations of Escherichia coli in the large intestine of conventional and gnotobiotic rats. Microb. Ecol. Health Dis. 7:263-273. 135 Neurath, A.R., S. Jiang, N. Strick, K. Lin, Y.-Y. Li and A.K. Debnath. 1996 Bovine -lactoglobulin modified by 3-hydroxy anhydride blocks the CD4 cell receptor for HIV. Nat. Med. 2:230-234. 136 Nyvad, B. and O. Fejerskov. 1986 Formation, composition and ultrastructure of microbial depositions on the tooth surface. p. 56-73. In: A. Thylstrup and O. Fejerskov (eds.) Textbook of cariology. Munksgaard. Copenhagen. 137 O’Sullivan, M.G., G. Thornton, G.C. O’Sullivan and J.K. Collins. 1992 Probiotic bacteria: myth or reality? Trends Food Sci. Technol. 3:309-314. 138 Ofek, I. and R.J. Doyle. 1994 Bacterial adhesion to cells and tissues. Chapman and Hall. London. 139 Ofek, I. and N. Sharon. 1996 Anti-Escherichia coli adhesin activity of cranberry and blueberry juices. Proc. Bat-Sheva seminar: Toward 63 140 141 142 143 144 145 146 147 148 149 150 64 anti-adhesion therapy of microbial diseases. p. 32-33. Zichron Yaakov, Israel. Ono, E., K. Abe, M. Nakazawa and M. Naiki. 1989 Ganglioside Epitope recognized by K99 fimbriae from enterotoxigenic Escherichia coli. J. Bact. 57:907-911. Parkkinen, J., G.N. Rogers, T.K. Korhonen, W. Dahr and J. Finne. 1986 Identification of the O-linked sialyloligosaccharides of glycophorin A as the erythrocyte receptors for S-fimbriated Escherichia coli. Infect. Immun. 54:37-42. Pearson, J.P., A. Allen and S. Parry. 1981 A 70 000-molecularweight protein isolated from purified pig gastric mucus glycoprotein by reduction of disulphide bridges and its implication in the polymeric structure. Biochem. J. 197:155-162. Pedersen, J., P. Klemm and W. Gaastra. 1986 987P fimbriae from porcine enterotoxigenic Escherichia coli characterization, N-terminal amino acid sequences and immunization with purified antigen. FEMS Microbiol. Lett. 66:229-234. Petschow, B.W. and R.D. Talbott. 1991 Response of Bifidobacterium species to growth promoters in human and cow milk. Ped. Res. 29:208-213. Pieroni, P., E.A. Worobec, W. Paranchych and G.D. Armstrong. 1988 Identification of a human erythrocyte receptor for colonization factor antigen I pili expressed by H10407 enterotoxigenic Escherichia coli. Infect. Immun. 56:1334-1340. Pothoulakis, C., C.P. Kelly, M.A. Joshi, N. Gao, C.J. O’Keane, I. Castagliuolo and J.T. Lamont. 1993 Saccharomyces boulardii inhibits Clostridium difficile toxine A binding and enterotoxicity in rat ileum. Gastroenterol. 104:1108-1115. Prescott, L.M., J.P. Harley and D.A. Klein. 1993 Microbiology. Wm.C. Brown Publishers. Dubuque. Pruitt, K.M., J. Tenovuo, B. Mansson-Rahemtulla, P. Harrington and D.C. Baldone. 1986 Is thiocyanate peroxidation at equilibrium in vivo? Biochim. Biophys. Acta 870:384-391. Purkayastha, S., C.V. Rao and M.E. Lamm. 1979 Structure of the carbohydrate chain of free secretory component from human milk. J. Biol. Chem. 254:6583-6587. Pærregaard, A., F. Espersen, O.M. Jensen and M. Skurnik. 1991 Interactions between Yersinia enterocolitica and rabbit ileal mucus: Growth, adhesion, penetration and subsequent changes in surface hydrophobicity and ability to adhere to ileal brush border membrane vesicles. Infect. Immun. 59:253-260. 151 Reid, G., R.L. Cook and A.W. Bruce. 1987 Examination of strains of lactobacilli for the properties that may influence bacterial interference in the urinary tract. J. Urol. 138:330-335. 152 Reid, G. and J.D. Sobel. 1987 Bacterial adherence in the pathogenesis of urinary tract infection: a review. Rev. Infect. Dis. 9:470-487. 153 Rest, R.F. 1995 Association of bacteria with human phagocytes. Methods Enzymol. 253:12-26. 154 Roberton, A.M., M. Mantle, R.E.F. Fahim, R.D. Specian, A. Bennick, S. Kawagashi, P. Sherman and J.F. Forstner. 1989 The putative ‘link’ glycopeptide associated with mucus glycoproteins. Biochem. J. 261:637-647. 155 Rose, M.C., W.A. Voter, C.F. Brown and B. Kaufman. 1984 Structural features of human tracheobronchial mucus glycoprotein. Biochem. J. 222:371-377. 156 Sajjan, S.U. and J.F. Forstner. 1990 Role of the putative “link” glycopeptide of intestinal mucin in binding of piliated Escherichia coli serotype O157:H7 strain CL-49. Infect. Immun. 58:868-873. 157 Salminen, S.J., Deighton, M. and S. Gorbach. 1993 Lactic acid bacteria in health and disease. p. 199-225. In: S. Salminen and A. von Wright (eds.) Lactic acid bacteria. Marcel Dekker Inc. New York. 158 Saloff-Coste, C.J. 1996 Health benefits of lactic acid bacteria. Danone World Newsletter 6. 159 Salyers, A.A. and D.D. Whitt. 1994 Bacterial pathogenesis: a molecular approach. ASM Press, Washington, D.C. 160 Sanchez, R., L. Kanarek, J. Koninkx, H. Hendriks, P. Lintermans, A. Bertels, G. Charlier and E. van Driesche. 1993 Inhibition of adhesion of enterotoxigenic Escherichia coli cells expressing F17 fimbriae to small intestinal mucus and brush-border membranes of young calves. Microb. Path. 15:407-419. 161 Sanders, M.E. 1993 Effect of consumption of lactic cultures on human health. Adv. Food Nutr. Res. 37:67-130. 162 Sandkvist, M., L.J. Overbye, T.K. Sixma, W.G.J. Hol and M. Bagdasarian. 1994 Assembly of Escherichia coli heat-labile enterotoxine and its secretion from Vibrio cholerae. P. 293-309. In: C.I. Kado and J.H. Crosa (eds.) Molecular mechanisms of bacterial virulence. Kluwer Academic Publishers. Dordrecht. 163 Sanford, P.A. 1992 Digestive system physiology. Edward Arnold, London. 164 Savage, D.C. 1977 Microbial ecology of the gastrointestinal tract. Ann. Rev. Microbiol. 3:107-133. 165 Savage, D.C. 1986 Gastrointestinal microflora in mammalian nutrition. Ann. Rev. Nutr. 6:155-178. 65 166 Savage, D.C. 1989 The normal human microflora-composition. p. 318. In: R. Grubb, T. Midtvedt and E. Norin (eds.) The regulatory and protective role of the normal microflora. M. Stockton Press, New York. 167 Schlager, T.A. and R.L. Guerrant. 1988 Seven possible mechanisms for Escherichia coli diarrhea. Infect. Dis. Clin. N. Am. 2:607-624. 168 Schlager, T.A., C. A. Wanke and R.L. Guerrant. 1990 Net fluid secretion and immaired villous function induced by colonization of the small intestine by nontoxigenic colonizing E. coli. Infect. Immun. 58:1337-1343. 169 Schroten, H., F.G. Hanisch, R. Plogmann, J. Hacker, G. Uhlenbruck, R. Nobisch-Bosch and V. Wahn. 1992 Inhibition of adhesion of S-fimbriated Escherichia coli to buccal epithelial cells by human milk fat globule membrane components: a novel aspect of the protective function of mucins in the nonimmunoglobulin fraction. Infect. Immun. 60:2893-2899. 170 Schroten, H., A. Lethen, F.G. Hanisch, R. Plogmann, J. Hacker, R. Nobisch-Bosch and V. Wahn. 1992 Inhibition of adhesion of Sfimbriated Echerichia coli to epithelial cells by meconium and faeces of breast-fed and formula-fed newborns: mucins are the major inhibitory component. J. Ped. Gastroenterol. Nutr. 15:150-158. 171 Schroten, H., R. Plogmann, F.G. Hanisch, J. Hacker, R. NobisBosch and V. Wahn. 1993 Inhibition of adhesion of S-fimbriated E. coli to buccal epithelial cells by human skim milk is predominantly mediated by mucins and depends on the period of lactation. Acta Paediatr. 62:6-11. 172 Sellwood, R., R.A. Gibbons, G.W. Jones and J.M. Rutter. 1975 Adhesion of enteropathogenic Escherichia coli to pig intestinal brush borders: the existence of two phenotypes. J. Med. Microbiol. 8:405-411. 173 Sheehan, J.K., K. Oats and I. Carlstedt. 1986 Electron microscopy of cervical, gastric and bronchial mucus glycoproteins. Biochem. J. 239:147-153. 174 Sherman, P., F. Cockerill III, R. Soni and J. Brunton. 1991 Outermembranes are competitive inhibitors of Escherichia coli O157:H7 adherence to epithelial cells. Infect. Immun. 59:890-899. 175 Shimizu, M., K. Yamauchi, Y. Miyauchi, T. Sakurai, Tokugawa, K. and R.A.J. McIlhinney. 1986 High-Mr glycoprotein profiles in human milk serum and fat-globule membrane. Biochem. J. 233:725-730. 176 Sjöbring, U., G. Pohl and A. Olsén. 1994 Plasminogen, absorbed by Escherichia coli expressing curli or by Salmonella enteritidis expressing thin aggregative fimbriae, can be activated by 66 177 178 179 180 181 182 183 184 185 186 187 188 189 190 simultaneously captured tissue-type plasminogen activator (t-PA). Mol. Microbiol. 14:443-452. Sjöstedt, S. 1989 The upper gastrointestinal microflora in relation to gastric diseases and gastric surgery. Acta Chir. Scand. Suppl. 551:157. Slomiany, B.L. and A. Slomiany. 1984 Lipids of mucous secretions of the alimentary tract. p.23-31. In: E.C. Boedeker (ed.) Attachment of organisms to the gut mucosa. Vol. II. CRC Press. Boca Raton. Slomiany, A. and B.L. Slomiany. 1996 Synthesis, structure and function of gastrointestinal mucin. Mucus Dialogue On-line 5:1-18 Smit, H., W. Gaastra, J.P. Kamerling, J.F.G. Vliegenthart and F.K. de Graaf. 1984 Isolation and structural characterization of the equine erythrocyte receptor for enterotoxigenic Escherichia coli K99 fimbrial adhesin. Infect. Immun. 46:578-584. Smith, B.F. and J.T. LaMont. 1984 Hydrophobic binding properties of bovine gallbladder mucin. J. Biol. Chem. 259:12170-12177. Sonstein, S.A. and J.C. Burnham. 1993 Effect of low concentrations of quinolone antibiotics on bacterial virulence mechanisms. J. Diagn. Microbiol. Infect. Dis. 16:277-289. Straube, E., G. Schmidt, R. Marre and J. Hacker. 1993 Adhesion and internalization of E. coli strains expressing various pathogenicity determinants. Zbl. Bakt. 278:218-228. Stryer, L. 1995 Biochemistry. W.H. Freeman and Company, New York. Stuart, D.I., K.R. Acharya, N.P.C. Walker, S.G. Smith, M. Lewis and D.C. Phillips. 1986 -lactalbumin possesses a novel calcium binding loop. Nature 324:84-87. Sutton, A.L., J.A. Patterson, A.B. Scheidt, D.T. Kelly, A.G. Mathew and K.A. Meyerholtz. 1994 Carbohydrate compounds to control enteropathogenic E. coli and intestinal VFA in the weanling pig. Proc. VIth International symposium on digestive physiology in pigs. p. 255-258. Bad Doberan, Germany. Tannock, G.W. 1981 Microbial interference in the gastrointestinal tract. ASEAN J. Clin. Sci. 2:2-34. Tannock, G.W. 1995 Normal microflora: An introduction to microbes inhabiting the human body. Chapman and Hall, London. Teneberg, S.T., P. Willemsen, F.K. de Graaf and K.-A. Karlsson. 1990 Receptor-active glycolipids of epithelial cells of the small intestine of young and adult pigs in relation to susceptibility to infection with Escherichia coli K99. FEBS Lett. 263:10-14. Teraguchi, S., K. Shin, Y. Fukuwatari and S. Shimamura. 1996 Glycans of bovine lactoferrin function as receptors for the type 1 fimbrial lectin of Echerichia coli. Infect. Immun. 64:1075-1077. 67 191 Timpte, C.S., A.E. Eckhardt, J.L. Abernethy and R.L. Hill. 1988 Porcine submaxillary gland apomucin contains tandemly repeated identical sequences of 81 residues. J. Biol. Chem. 263:1081-1088. 192 Turnberg, L.A. 1988 Pathophysiology of diarrhoea in enteric infections. Baillièr’s Clin. Trop. Med. Communicable Dis. 3:391-400. 193 Vázquez-Juárez, R., T. Andlid and L. Gustafsson. 1994 Cell surface hydrophobicity and its relation to adhesion of yeasts isolated from fish gut. Colloids Surfaces B: Biointerfaces. 2:199-208. 194 Verwey, E.J. and J.T.G. Overbeek. 1948 On the interaction of spherical colloidal particles. p. 135-185. In: Theory of the stability of lyophobic colloids. Part III, Elsevier, Amsterdam. 195 Wadström, T. 1988 Adherence traits and mechanisms of microbial adhesion in the gut. Baillière’s Clin. Trop. Med. Communicable Dis. 3:417-433. 196 Weiser, M.M. 1984 The synthesis of intestinal glycoproteins with special reference to vitamin D-dependent processes, attachment of microorganisms, and membrane shedding. p. 89-98 In: E.C. Boedeker (ed.) Attachment of organisms to the gut mucosa. Vol. II. CRC Press. Boca Raton. 197 Willemsen, P.T.J. and F.K. de Graaf 1992 Age and serotype dependent binding of K88 fimbriae to porcine intestinal receptors. Microbial Path. 12:367-375. 198 Wold, A.E., M. Thorssén, S. Hull and C. Svanborg-Edén. 1988 Attachment of Escherichia coli via mannose or GalGalcontaining receptors to human colonic epithelial cells. Infect. Immun. 56:2531-2537. 199 Yamauchi, K., M. Tomita, T.J. Giehl and R.T. Ellison III. 1993 Antibacterial activity of lactoferrin and a pepsin-derived lactoferrin peptide fragment. Infect. Immun. 61:719-728. 200 Zafriri, D., I. Ofek, R. Adar, M. Pocino and N. Sharon. 1989 Inhibitory activity of cranberry juice on adherence of type 1 and type P fimbriated Escherichia coli to eucaryotic cells. Antimicrob. Agents Chemother. 33:92-98. 201 Zobel, C.E. 1943 The effect of solid surfaces upon bacterial activity. J. Bacteriol. 46:39-56. 68