Microbial biofilms and gastrointestinal diseases.

The majority of bacteria live not planktonically, but as residents of sessile biofilm communities. Such populations have been defined as 'matrix-enclosed microbial accretions, which adhere to both biological and nonbiological surfaces'. Bacterial formation of biofilm is implicated in many chronic disease states. Growth in this mode promotes survival by increasing community recalcitrance to clearance by host immune effectors and therapeutic antimicrobials. The human gastrointestinal (GI) tract encompasses a plethora of nutritional and physicochemical environments, many of which are ideal for biofilm formation and survival. However, little is known of the nature, function, and clinical relevance of these communities. This review summarizes current knowledge of the composition and association with health and disease of biofilm communities in the GI tract.

Effects of antibiotics on bacterial species composition and metabolic activities in chemostats containing defined populations of human gut microorganisms.

The composition and metabolic activities of the human colonic microbiota are modulated by a number of external factors, including diet and antibiotic therapy. Changes in the structure and metabolism of the gut microbiota may have long-term consequences for host health. The large intestine harbors a complex microbial ecosystem comprising several hundreds of different bacterial species, which complicates investigations on intestinal physiology and ecology. To facilitate such studies, a highly simplified microbiota consisting of 14 anaerobic and facultatively anaerobic organisms (Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium pseudolongum, Bifidobacterium adolescentis, Clostridium butyricum, C. perfringens, C. bifermentans, C. innocuum, Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Lactobacillus acidophilus) was used in this investigation. Ampicillin [9.2 µg (ml culture)(-1)] was added to two chemostats operated at different dilution rates (D; 0.10 h(-1) and 0.21 h(-1)), and metronidazole [76.9 µg (ml culture)(-1)] was added to a third vessel (D = 0.21 h(-1)). Perturbations in bacterial physiology and metabolism were sampled over a 48-h period. Lactobacillus acidophilus and C. bifermentans populations did not establish in the fermentors under the imposed growth conditions. Ampicillin resulted in substantial reductions in bacteroides and C. perfringens populations at both dilution rates. Metronidazole strongly affected bacteroides communities but had no effect on bifidobacterial communities. The bacteriostatic effect of ampicillin on bifidobacterial species was growth rate dependent. Several metabolic activities were affected by antibiotic addition, including fermentation product formation and enzyme synthesis. The growth of antibiotic-resistant bifidobacteria in the large bowel may enable them to occupy ecological niches left vacant after antibiotic administration, preventing colonization by pathogenic species.

Longitudinal analyses of gut mucosal microbiotas in ulcerative colitis in relation to patient age and disease severity and duration.

Bacteria belonging to the normal colonic microbiota are associated with the etiology of ulcerative colitis (UC). Although several mucosal species have been implicated in the disease process, the organisms and mechanisms involved are unknown. The aim of this investigation was to characterize mucosal biofilm communities over time and to determine the relationship of these bacteria to patient age and disease severity and duration. Multiple rectal biopsy specimens were taken from 33 patients with active UC over a period of 1 year. Real-time PCR was used to quantify mucosal bacteria in UC patients compared to 18 noninflammatory bowel disease controls, and the relationship between indicators of disease severity and bacterial colonization was evaluated by linear regression analysis. Significant differences were detected in bacterial populations on the UC mucosa and in the control group, which varied over the study period. High clinical activity indices (CAI) and sigmoidoscopy scores (SS) were associated with enterobacteria, desulfovibrios, type E Clostridium perfringens, and Enterococcus faecalis, whereas the reverse was true for Clostridium butyricum, Ruminococcus albus, and Eubacterium rectale. Lactobacillus and bifidobacterium numbers were linked with low CAI. Only E. rectale and Clostridium clostridioforme had a high age dependence. These findings demonstrated that longitudinal variations in mucosal bacterial populations occur in UC and that bacterial community structure is related to disease severity.

Bacteria, colonic fermentation, and gastrointestinal health.

The colonic microbiota plays an important role in human digestive physiology and makes a significant contribution to homeostasis in the large bowel. The microbiome probably comprises thousands of different bacterial species. The principal metabolic activities of colonic microorganisms are associated with carbohydrate and protein digestion. Nutrients of dietary and host origin support the growth of intestinal organisms. Short-chain fatty acids (SCFAs), predominantly acetate, propionate, and butyrate, are the principal metabolites generated during the catabolism of carbohydrates and proteins. In contrast, protein digestion yields a greater diversity of end products, including SCFAs, amines, phenols, indoles, thiols, CO2, H2, and H2S, many of which have toxic properties. The majority of SCFAs are absorbed from the gut and metabolized in various body tissues, making a relatively small but significant contribution to the body's daily energy requirements. Carbohydrate fermentation is, for the most part, a beneficial process in the large gut, because the growth of saccharolytic bacteria stimulates their requirements for toxic products associated with putrefaction, for incorporation into cellular proteins, thereby protecting the host. However, as digestive materials move along the gut, carbohydrates become depleted, which may be linked to the increased prevalence of colonic disease in the distal bowel.

Effect of a synbiotic on microbial community structure in a continuous culture model of the gastric microbiota in enteral nutrition patients.

Patients with dysphagia require long-term nutritional support. This can be delivered by the enteral route via a percutaneous endoscopic gastrostomy (PEG) tube. Enteral nutrition (EN) bypasses the body's innate defences that prevent the microbial colonization of the proximal gut, which predisposes to microbial overgrowth. A continuous culture model simulating the upper gastrointestinal tract microbiota of EN patients was used to investigate the effects of a synbiotic (Lactobacillus acidophilus DUN-311, Bifidobacterium bifidum BB-02, Bifidobacterium lactis BL-01, Synergy 1) on microbial community structure and metabolism. A PEG tube was inserted into the fermenters to study biofilm formation. The synbiotic delivered in sterile semi-skimmed milk (SSSM) was introduced either 48 h prior to or after PEG tube insertion. The synbiotic reduced biofilm formation on PEG tube surfaces, with suppression of Escherichia coli and Klebsiella pneumoniae when it was added subsequent to PEG insertion. When synbiotic feeding was commenced prior to PEG insertion, colonization by Staphylococcus aureus, Candida albicans and Candida famata was also inhibited. Lactate production increased in response the synbiotic or control (SSSM). These results indicate that the use of a synbiotic has the potential to reduce pathogen colonization on PEG tube surfaces in vivo, thereby reducing the incidence of biofilm-related infectious complications.

Bacterial translocation in cirrhosis is not caused by an abnormal small bowel gut microbiota.

Sepsis is common in liver cirrhosis, and animal studies have shown the gut to be the principal source of infection, through bacterial overgrowth and translocation in the small bowel. A total of 33 patients were recruited into this study, 10 without cirrhosis and 23 with cirrhotic liver disease. Six distal duodenal biopsies were obtained and snap frozen for RNA and DNA extraction, or frozen for FISH. Peripheral venous bloods were obtained from 30 patients, including 17 chronic liver disease patients. Samples were analysed by real-time PCR, to assess total bacteria, bifidobacteria, bacteroides, enterobacteria, staphylococci, streptococci, lactobacilli, enterococci, Helicobacter pylori and moraxella, as well as TNF-α, IL-8 and IL-18. There was no evidence of bacterial overgrowth with respect to any of the individual bacterial groups, with the exception of enterococci, which were present in higher numbers in cirrhotic patients (P = 0.04). There were no significant differences in any of the cytokines compared to the controls. The small intestinal mucosal microbiota in cirrhotic patients was qualitatively and quantitatively normal, and this shifts the focus of disease aetiology to factors that reduce gut integrity, failure of mechanisms to remove translocating bacteria, or the large bowel as the source of sepsis.

Fermentation in the human large intestine: its physiologic consequences and the potential contribution of prebiotics.

The human large intestine harbors a complex microbiota containing many hundreds of different bacterial species. Although structure/function relationships between different components of the microbiota are unclear, this complex multicellular entity plays an important role in maintaining homeostasis in the body. Many of the physiologic properties of the microbiota can be attributed to fermentation and the production of short-chain fatty acids (SCFAs), particularly acetate, propionate, and butyrate. In healthy people, fermentation processes are largely controlled by the amounts and different types of substrate, particularly complex carbohydrates that are accessible to bacteria in the colonic ecosystem. However, other factors impact on bacterial metabolism in the large gut, including large bowel transit time, the availability of inorganic terminal electron acceptors, such as nitrate and sulfate, and gut pH. They all affect the types and levels of SCFA that can be formed by the microbiota. This is important because to a large extent, acetate, propionate, and butyrate have varying physiologic effects in different body tissues. Prebiotics such as galactooligosaccharides together with inulins and their fructooligosaccharide derivatives have been shown to modify the species composition of the colonic microbiota, and in various degrees, to manifest several health-promoting properties related to enhanced mineral absorption, laxation, potential anticancer properties, lipid metabolism, and anti-inflammatory and other immune effects, including atopic disease. Many of these phenomena can be linked to their digestion and SCFA production by bacteria in the large gut.

Recommendations for probiotic use-2011 update.

This study describes the consensus opinion of the participants of the third Yale Workshop on probiotic use. There were 10 experts participating. The recommendations update those of the first 2 meetings that were published in 2005 and 2008. The workshop presentations and papers in this supplement relate to the involvement of normal microbiota involved in intestinal microecology, how the microbes interact with the intestine to affect our immunologic responses, the stability and natural history of probiotic organisms, and the role of the intestinal microbatome with regard to affecting cardiac risk factors and obesity. Recommendations for the use of probiotics in necrotizing enterocolitis, childhood diarrhea, inflammatory bowel disease, irritable bowel syndrome, and Clostridium difficile diarrhea are reviewed. As in previous publications, the recommendations are given as A, B, or C ratings. The recent positive experiences with bacteriotherapy (fecal microbiome transplant) are also discussed in detail and a positive recommendation is made for use in severe resistant C. difficile diarrhea.

Mucosal biofilm communities in the human intestinal tract.

Complex and highly variable site-dependent bacterial ecosystems exist throughout the length of the human gastrointestinal tract. Until relatively recently, the majority of our information on intestinal microbiotas has come from studies on feces, or from aspirates taken from the upper gut. However, there is evidence showing that mucosal bacteria growing in biofilms on surfaces lining the gut differ from luminal populations, and that due to their proximity to the epithelial surface, these organisms may be important in modulating the host's immune system and contributing to some chronic inflammatory diseases. Over the past decade, increasing interest in mucosal bacteria, coupled with advances in molecular approaches for assessing microbial diversity, has begun to provide some insight into the complexity of these mucosa-associated communities. In gastrointestinal conditions such as inflammatory bowel diseases (ulcerative colitis, Crohn's disease), it has been shown that a dysbiosis exists in microbial community structure, and that there is a reduction in putatively protective mucosal organisms such as bifidobacteria. Therefore, manipulation of mucosal communities may be beneficial in restoring normal functionality in the gut, thereby improving the immune status and general health of the host. Biofilm structure and function has been studied intensively in the oral cavity, and as a consequence, mucosal communities in the mouth will not be covered in this chapter. This review addresses our current knowledge of mucosal populations in the gastrointestinal tract, changes that can occur in community structure in disease, and therapeutic modulation of biofilm composition by antibiotics, prebiotics, and probiotics.

Adherence and cytokine induction in Caco-2 cells by bacterial populations from a three-stage continuous-culture model of the large intestine.

Adherence of bacteria to epithelial cells is an important step in colonization and immune modulation in the large bowel. The aims of this study were to use a three-stage continuous-culture system (CCS) to investigate how environmental factors affect bacterial attachment to Caco-2 cells and modulation of cytokine expression by gut microorganisms, including a probiotic Bifidobacterium longum strain, DD2004. The CCS simulated environmental conditions in the proximal large intestine (vessel 1 [V1]) and distal colon (V2 and V3) at two different system retention times (R) within the range of normal colonic transits (20 and 60 h). The model was inoculated with human fecal material, and fluorescence in situ hybridization (FISH) was used to characterize microbial populations and to assess bacterial attachment to Caco-2 cells. Real-time quantitative PCR (qPCR) was employed to measure cytokine gene expression following challenge with bacteria from different components of the CCS in the presence and absence of B. longum. At an R of 60 h, bacterial adherence increased from V1 to V3, but this trend was reversed at an R of 20 h. Atopobia were the predominant adherent organisms detected at both system retention times in each culture vessel. Modulation of transforming growth factor ß1 (TGF-ß1), interleukin 6 (IL-6), and IL-18 gene expression by CCS bacteria was marked at an R of 60 h, while at an R of 20 h, IL-4, IL-10, TGF-ß2, IL-1α, and tumor necrosis factor alpha (TNF-α) were significantly affected. The addition of B. longum affected cytokine expression significantly at both retention times. This study demonstrates that environmental determinants regulate the adherence properties of intestinal bacteria and their abilities to regulate cytokine synthesis.

Intestinal bacteria and inflammatory bowel disease.

Crohn's disease (CD) and ulcerative colitis (UC) are the two principal forms of inflammatory bowel disease (IBD). Animal studies show that bacteria are involved in the etiology of IBD, and much is now known about the inflammatory processes associated with CD and UC, as well as the underlying genetic, environmental, and lifestyle issues that can affect an individual's predisposition to these diseases. However, while a number of candidate microorganisms have been put forward as causative factors in IBD, the primary etiologic agents are unknown. This review discusses the potential role of luminal and mucosal microbial communities in the etiology of IBD, and outlines studies that have been made using a variety of biotherapeutic therapies, involving the use of antibiotics, probiotics, prebiotics, and synbiotics.

Prebiotics, synbiotics and inflammatory bowel disease.

The normal colonic microflora is intimately involved in the aetiology of inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD). These conditions are often refractile to conventional treatments involving the employment of anti-inflammatory and immunosuppressant drugs, and this has led to a search for alternative therapies based on the use of probiotics, prebiotics and synbiotics. The majority of investigations in this area have been done with probiotics, and while there is increasing interest in the abilities of prebiotics and synbiotics to control the symptoms of IBD, very few randomised controlled trials have been reported. Although the results have been variable, human and animal studies have demonstrated that in many circumstances, these functional foods can alter the composition of the colonic microbiota, reduce inflammatory processes in the gut mucosa, and have the potential to induce disease remission. More work is needed to understand the effects of prebiotics and synbiotics on microbial communities in the gut, and their interactions with the host's immune system.

Mucosa-associated bacterial diversity in relation to human terminal ileum and colonic biopsy samples.

Little is known about bacterial communities that colonize mucosal surfaces in the human gastrointestinal tract, but they are believed to play an important role in host physiology. The objectives of this study were to investigate the compositions of these populations in the distal small bowel and colon. Healthy mucosal tissue from either the terminal ileum (n = 6) or ascending (n = 8), transverse (n = 8), or descending colon (n = 4) of 26 patients (age, 68.5 +/- 1.2 years [mean +/- standard deviation]) undergoing emergency resection of the large bowel was used to study these communities. Mucosa-associated eubacteria were characterized by using PCR-denaturing gradient gel electrophoresis (DGGE), while real-time PCR was employed for quantitative analysis. Mucosal communities were also visualized in situ using confocal laser scanning microscopy. DGGE banding profiles from all the gut regions exhibited at least 45% homology, with five descending colon profiles clustering at ca. 75% concordance. Real-time PCR showed that mucosal bacterial population densities were highest in the terminal ileum and that there were no significant differences in overall bacterial numbers in different parts of the colon. Bifidobacterial numbers were significantly higher in the large bowel than in the terminal ileum (P = 0.006), whereas lactobacilli were more prominent in the distal large intestine (P = 0.019). Eubacterium rectale (P = 0.0004) and Faecalibacterium prausnitzii (P = 0.001) were dominant in the ascending and descending colon. Site-specific colonization in the gastrointestinal tract may be contributory in the etiology of some diseases of the large intestine.

Microbial colonization of the upper gastrointestinal tract in patients with Barrett's esophagus.

BACKGROUND: Barrett's esophagus (BE) is a complication of chronic gastroesophageal reflux disease, in which patients are at greatly increased risk of esophageal dysplasia and adenocarcinoma. Over the past 2 decades, there has been an increase in the incidence of both BE and adenocarcinoma; however, the involvement of microorganisms in BE is uncertain. The aim of this study was to characterize microbial communities in esophageal aspirate specimens and on distal esophageal mucosal samples from patients with BE. METHODS: Biopsy and aspirate specimens were obtained by endoscopic examination from 7 patients with BE and 7 control subjects without BE. Samples were cultured under aerobic, anaerobic, and microaerophilic conditions for yeasts and bacteria, including Helicobacter pylori. Bacterial isolates were identified by 16S ribosomal RNA gene sequencing. Fluorescence microscopic examination was also used to determine the spatial localization of these organisms on mucosal surfaces. Significant colonization was detected in 6 patients with BE and in 4 control subjects. RESULTS: Overall, 46 bacterial species belonging to 16 genera were detected, with 10 species being common in both groups. Both aspirate and biopsy samples from patients with BE contained complex populations of bacteria. Uniquely, high levels of Campylobacter species (Campylobacter concisus and Campylobacter rectus), which have been linked to enteritis, periodontal infections, and tumor formation in animals, were found in 4 (57%) of 7 patients with BE but in none of the control subjects. Microscopic examination revealed that bacteria on mucosal biofilms often occurred in microcolonies. CONCLUSIONS: The occurrence of nitrate-reducing Campylobacter species in patients with BE may suggest that there is a link in either the initiation, maintenance, or exacerbation of disease processes leading to adenocarcinoma formation.

Lactobacillus gasseri Gasser AM63(T) degrades oxalate in a multistage continuous culture simulator of the human colonic microbiota.

Colonic oxalate-degrading bacteria have been shown to play an important role in human kidney stone formation. In this study, molecular analysis of the Lactobacillus gasseri genome revealed a cluster of genes encoding putative formyl coenzyme A transferase (frc) and oxalyl coenzyme A decarboxylase (oxc) homologues, possibly involved in oxalate degradation. The ability of Lactobacillus gasseri Gasser AM63(T) to degrade oxalate was confirmed in vitro. Transcription of both genes was induced by oxalate, and reverse transcription-PCR confirmed that they were co-transcribed as an operon. A three-stage continuous culture system (CCS) inoculated with human fecal bacteria was used to model environmental conditions in the proximal and distal colons, at system retention times within the range of normal colonic transit rates (30 and 60 hours). A freeze-dried preparation of L. gasseri was introduced into the CCS under steady-state growth conditions. Short chain fatty acid analysis indicated that addition of L. gasseri to the CCS did not affect the equilibrium of the microbial ecosystem. Oxalate degradation was initiated in the first stage of the CCS, corresponding to the proximal colon, suggesting that this organism may have potential therapeutic use in managing oxalate kidney stone disease in humans.

Models for intestinal fermentation: association between food components, delivery systems, bioavailability and functional interactions in the gut.

There is increasing interest in the human colonic microbiota and in the way its metabolic activities impact on host health and well-being. For most practical purposes, however, the large bowel is inaccessible for routine investigation, and a variety of animal and in vitro model systems have been developed to study the microbiota. In vitro models range from simple closed systems using pure or defined mixed populations of bacteria, or faecal material, to more sophisticated complex multistage continuous cultures that are able to simulate many of the spatial, temporal and environmental attributes that characterize microbiological events in different regions of the large gut. Recent developments using these systems have enabled modelling of surface colonisation and biofilm development, a hitherto neglected area of study.

Composition and metabolic activities of bacterial biofilms colonizing food residues in the human gut.

Bacteria growing in the human large intestine live in intimate association with the host and play an important role in host digestive processes, gut physiology, and metabolism. Fecal bacteria have been investigated extensively, but few studies have been done on biofilms that form on digestive wastes in the large bowel. The aims of this investigation were to investigate the composition and metabolic activities of bacterial communities that colonize the surfaces of food residues in fecal material, with respect to their role in the fermentation of complex carbohydrates. Fresh stools were obtained from 15 healthy donors, and food residues were separated by filtration. Adherent bacteria were removed by surfactant treatment for microbiological analysis and fermentation studies. Scanning electron microscopy and fluorescent in situ hybridization in conjunction with confocal laser scanning microscopy (CLSM) were used to visualize intact biofilms. Results showed that bacterial populations strongly adhering to particulate matter were phenotypically similar in composition to unattached communities, with bacteroides and bifidobacteria predominating. Biofilms comprised a mixture of living and dead bacteria, and CLSM showed that bifidobacteria in the biofilms occurred as isolated dispersed cells and in microcolonies near the interface with the substratum. Fermentation experiments with a variety of complex carbohydrates demonstrated that biofilm populations were more efficient in digesting polysaccharides, while nonadhering communities fermented oligosaccharides most rapidly. Acetate was the principal fermentation product formed by biofilm bacteria, whereas higher levels of butyrate were produced by nonadherent populations, showing that the two communities were metabolically distinct.

Studies on the effect of system retention time on bacterial populations colonizing a three-stage continuous culture model of the human large gut using FISH techniques.

Fluorescence in situ hybridization was used to quantitate bacteria growing in a three-stage continuous culture system inoculated with human faeces, operated at two system retention times (60 and 20 h). Twenty-three different 16S rRNA gene oligonucleotide probes of varying specificities were used to detect bacteria. Organisms belonging to genera Bacteroides and Bifidobacterium, together with the Eubacterium rectale/Clostridium coccoides group, the Atopobium, Faecalibacterium prausnitzii and Eubacterium cylindroides groups, as well as the segmented filamentous bacteria, the Roseburia intestinalis group and lactic acid bacteria, were all present in high numbers in the continuous culture system. Other groups and species such as Ruminococci and Enterobacteria also persisted in the model, though not always at levels that allowed reliable quantitation. Some organisms such as Streptococci and Corynebacteria, present in the faecal inoculum, did not colonize the system. Other probes specific for Eubacterium lentum and for members of the genus Desulfovibrio did not detect these organisms at any time. Short chain fatty acid production was always highest in vessel I of the continuous culture system, however, a marked increase in acetate formation and a reduction in butyrate production occurred when system retention time was reduced to 20 h, which correlated with reductions in the numbers of butyrate-producing Roseburia.

Characterisation of intestinal bacteria in infant stools using real-time PCR and northern hybridisation analyses.

Real-time PCR and northern hybridisations were used to quantify bacterial populations in the large gut of infants. PCR primers for rapid, sensitive, high throughput detection of bifidobacteria, bacteroides, sulphate-reducing bacteria and Enterococcus faecalis, based on analysis of 16S rRNA genes were used. Bacterial populations were analysed in faeces from 40 infants aged 0-6, 7-12 and 13-24 months. The effects of breast versus bottle feeding was also investigated. Real-time PCR indicated that bacteroides and desulfovibrio numbers increased markedly in the 7-12 and 13-24 month age groups, and that the reverse occurred with Ent. faecalis. With the exception of desulfovibrios, this was seen with northern hybridisations, which also showed increased colonisation by the Clostridium coccoides group and Faecalibacterium prausnitzii after 6 months. Both methodologies indicated increased bifidobacteria in breast-fed babies, and higher levels of desulfovibrios in bottle-fed children.

Colonization of mucin by human intestinal bacteria and establishment of biofilm communities in a two-stage continuous culture system.

The human large intestine is covered with a protective mucus coating, which is heavily colonized by complex bacterial populations that are distinct from those in the gut lumen. Little is known of the composition and metabolic activities of these biofilms, although they are likely to play an important role in mucus breakdown. The aims of this study were to determine how intestinal bacteria colonize mucus and to study physiologic and enzymatic factors involved in the destruction of this glycoprotein. Colonization of mucin gels by fecal bacteria was studied in vitro, using a two-stage continuous culture system, simulating conditions of nutrient availability and limitation characteristic of the proximal (vessel 1) and distal (vessel 2) colon. The establishment of bacterial communities in mucin gels was investigated by selective culture methods, scanning electron microscopy, and confocal laser scanning microscopy, in association with fluorescently labeled 16S rRNA oligonucleotide probes. Gel samples were also taken for analysis of mucin-degrading enzymes and measurements of residual mucin sugars. Mucin gels were rapidly colonized by heterogeneous bacterial populations, especially members of the Bacteroides fragilis group, enterobacteria, and clostridia. Intestinal bacterial populations growing on mucin surfaces were shown to be phylogenetically and metabolically distinct from their planktonic counterparts.