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Spatial organization of intestinal microbiota in health and disease

Spatial organization of intestinal microbiota in health and disease
Authors:
Alexander Swidsinski, MD
Vera Loening-Baucke, MD
Section Editor:
J Thomas Lamont, MD
Deputy Editor:
Shilpa Grover, MD, MPH, AGAF
Literature review current through: Dec 2022. | This topic last updated: Mar 01, 2021.

INTRODUCTION — The alimentary tract represents an interface between the external environment and the body. Within it exists a complex polymicrobial ecology that interacts with the internal and external environment and has an important influence on health and disease.

The properties of isolated microorganisms do not explain how the polymicrobial community functions or why its organisms can grow under conditions that should be deadly to them [1]. An understanding of how microorganisms interact with each other, their host, and the luminal contents is expanding rapidly.

A major advance in understanding the function of the intestinal microbiota has been the development of techniques that permit a detailed assessment of the composition of the flora and its distribution throughout the alimentary tract. One of the methods to visualize single bacterial species within complex communities is called ribosomal RNA fluorescence in situ hybridization (FISH).

Each bacterium possesses tens of thousands of ribosomes, each of which includes a copy of the bacterial RNA. Some of the regions of the ribosomal RNA are strain-specific, others are universal for groups, domains, or even kingdoms. Synthetically produced oligonucleotides that are complementary to sequences of interest can be labeled with fluorescent dye and added to samples containing bacteria. These oligonucleotides, called FISH probes, hybridize with RNA of bacterial ribosomes. Bacteria can be visualized with the microscope directly without additional enhancement because of the high number of ribosomes within each bacterium [2,3].

The names of the FISH probes described in this topic review are based on abbreviations of probeBase online resource for rRNA-targeted oligonucleotide probes [4].

EVOLUTION'S ROLE IN THE DISTRIBUTION OF INTESTINAL MICROBIOTA — Animals have developed multiple mechanisms to control bacteria that enter the digestive tract, which can be broadly divided into those that suppress their growth (eg, salivary lysozyme, gastric acid, secretion of defensins) or those that separate them from the host by creating a physical barrier (eg, the mucus barrier). Polymicrobial communities such as exist in the intestine are much more resilient to these defenses than bacterial monocultures. They exhibit a coordinated response to environmental challenges, resist antibiotics, and can evade the host's immune response and are thus able to persist under extreme conditions.

As a result, the control of bacterial growth by the host is never absolute and the intestine is never sterile. The occurrence, composition, and organization of intestinal microbiota in each gut segment depend upon whether suppression or separation dominates. In gut regions with active suppression of microbiota, the bacteria are occasional, of variable composition, and of low concentration. A complete separation of bacteria from the mucosa and low levels of suppression lead to the development of an intestinal reservoir in which bacteria can grow and reach high concentrations. Bacteria are indigenous to these intestinal regions.

The balance of suppression and separation mechanisms depends upon the evolutionary impact of bacteria on promoting health or causing disease. As an example, plant-feeding animals use microorganisms to digest cellulose, which would otherwise be indigestible. Thus, bacteria that digest cellulose are indigenous to the rumen of these animals. On the other hand, the nutrients resulting from bacterial degradation in ruminates are resorbed in the small intestine where bacteria would be clear competitors and are therefore suppressed. Thus, bacteria are occasional and present in only low concentrations in the small intestine.

Similarly, bacteria in the colon are important for maintaining the health of colonic enterocytes and for digesting nutrients that could not be absorbed in the small bowel. As a result, bacteria exist in high concentrations in the colon. (See 'Bacteria in the colon' below.)

BACTERIA IN THE UPPER GASTROINTESTINAL TRACT

Mouth — Microorganisms from the external world first contact the alimentary tract in the mouth. Bacteria can be found in high concentrations on food remnants, the dental surface and within or attached to desquamated epithelial cells, and suspended in oral secretions. There are also high concentrations of bacteria in saliva [1].

Despite the high concentration of bacteria in the oral cavity, samples taken from the stratified epithelium of the mouth and saliva taken directly from the salivary duct are virtually sterile in healthy persons. This reflects defensive mechanisms working in concert to suppress bacterial adhesion and growth.

Tonsils — Similar to the rest of the oral cavity, no constant pattern of colonization can be found on the surface of the tonsillar epithelium [5]. Most of the epithelial surface of tonsils is free of bacteria even in tonsillectomy specimens obtained from individuals with chronic tonsillitis (picture 1). When bacteria were found, they were localized either to circumscribed regions of diffuse infiltration, within macrophages, superficial infiltrates, singular purulent fissures, or abscesses. The composition of bacteria within infectious tonsillar foci varies among individuals and often differs even between different regions of the same tonsil, indicating that bacteria are not indigenous to the tonsils but represent remnants of an incompletely cured purulent process.

Stomach and duodenum — Cultures of fluid from the stomach or duodenum of healthy volunteers contain bacterial concentrations of 10(3) to 10(4)/mL, depending in part upon the composition of digested food. Fluorescence in situ hybridization (FISH) demonstrates that bacteria in the stomach and duodenum are localized strictly within the intestinal lumen and separated from the mucosa by a mucus layer. Their composition is variable, reflecting the heterogeneous composition of the ingested flora. In healthy individuals, the mucosa and the mucus layer of the stomach and duodenum are free of bacteria. In patients with Helicobacter pylori infection, the mucosal surface of the stomach is covered with a bacterial biofilm in which H. pylori is predominant [6].

Pancreatic tract — The microflora of the normal pancreatic duct has not been extensively studied, since samples available from healthy individuals are not readily available. FISH analysis of pancreatic duct biopsies obtained during endoscopic retrograde cholangiopancreatography from patients with benign or malignant obstruction of the pancreatic duct have demonstrated islands of bacterial adhesion in approximately 70 percent of the biopsies [7]. The anatomically normal pancreatic duct epithelium is generally sterile. Thus, bacterial islands appear to be located in regions of disturbed duct anatomy. Bacterial species in the pancreatic duct are diverse and highly variable among individuals.

Biliary tract — Biopsies of healthy bile ducts are not readily available and thus the microflora of the biliary epithelium in health has not been extensively evaluated. Gallbladder resection specimens obtained from patients who did not receive preoperative antibiotics are free of bacteria, indicating that the bile duct epithelium is normally not colonized.

The situation is different in the presence of foreign bodies such as biliary stents (picture 2). The colonization of the biliary stents by polymicrobial biofilm can be documented within a week after implantation [7,8]. Both aerobic and anaerobic bacteria, which are commonly found in the intestine, can be identified. The microbial colonization starts with the distal end of the biliary stent and advances proximally. Bacteria are located mainly on the inner surface of plastic stents. By contrast, the surface of biliary stents facing normal epithelium is not colonized, indicating that the healthy epithelial layer efficiently resists microbial colonization [7].

The rapid development of biofilms at the inner surface of the biliary stents indicates that the biliary and pancreatic secretions alone are unable to prevent the development of bacterial biofilms on foreign bodies. Interestingly, the bacterial biofilm disappears as soon as the stent is occluded by sludge, suggesting that organic substances in sludge help prevent bacterial growth.

Gallstones — A similar trend of the bacterial biofilm vanishing after local deposition of organic substances can be observed in gallstones. Because gallstones are essentially foreign bodies, they should be permanently colonized. Bacteria can indeed be found in loose brown pigment stones and sludge, which are an initial stage in the formation of gallstones.

The natural history of the gallstone is a progression from brown to composite and then to cholesterol gallstones. The cholesterol stones can reach considerable sizes and persist in the human body over many decades. Nevertheless, cholesterol gallstones obtained after cholecystectomy are mostly sterile. This suggests that sedimentation of cholesterol and sludge within the bacterial biofilm is an integral part of some kind of protective mechanism against otherwise extremely recalcitrant infections [7]. The body's sedimentary mechanism as a defense appears extremely efficient in suppressing bacterial biofilms on foreign bodies when compared to the complete ineffectiveness of presently available antibiotics.

Small intestine — The epithelial surface of the small intestine in a healthy human is not colonized. Occasional groups of bacteria can be found in low concentrations of 10(5) or less within the lumen. Bacteria do not form conglomerates and spatial structures and the luminal contents are separated from the mucosa by a mucus layer.

The situation is similar in mice but not in rats. In most groups of wild-type rats, segmented filamentous bacteria (SFB) can be observed tightly attached to the epithelial surface and located between villi throughout the small intestine. The SFB adherence in rats is not accompanied by an increase in concentrations of other bacterial groups or leukocytes. It is difficult to say whether segmented filamentous bacteria are pathogens such as Helicobacter species, saprophytic or symbiotic bacteria with an unknown role, because of the high prevalence of adherent SFB in the small intestines of wild-type rats.

In humans, the adherence of segmented filamentous bacteria has not been observed either in health or disease. Pathologic conditions with altered microbiota in the small intestine include acute and chronic infections, bacterial overgrowth, and inflammatory bowel disease. Common to all of these conditions are a disruption of the mucus barrier, loss of bacterial separation between mucosa and lumen, bacterial adherence, invasion, and translocation [9]. (See 'Disruption of the mucus barrier' below.)

BACTERIA IN THE COLON

The role of microbiota in colonic function — The large intestine is a bioreactor in which the host uses bacteria to degrade indigestible leftovers. Bacteria produce valuable substances such as vitamins and short fatty acids by degrading waste products.

In humans, resorption in the large intestine is restricted mostly to water and electrolytes. Bacteria in the human colon are mainly responsible for the reduction of the fecal mass. Which bacterial species are responsible for these processes are not well known. However, a reasonable assumption is that the numerically predominant bacteria are indispensable for the biochemical processes that occur in the colon. Eubacterium rectale (Roseburia spp), Faecalibacterium prausnitzii, and Bacteroides groups comprise each 10 to 30 percent and cumulative 70 percent of the total microbiota in humans [10,11]. All other bacterial groups are present only in subgroups of individuals or in parts of the colon.

Although the fecal flora is one of the most well-characterized microbiota, many of the bacterial species inhabiting the large intestine are unknown. Strict anaerobic species predominate, although the diversity of bacteria is high and consists of approximately 3000 to 5000 species [12].

In the colon, bacteria reach concentrations of up to the 10(11)/mL and compose up to 90 percent of the fecal mass. Such high bacterial concentrations can be achieved only under active facilitation of bacterial growth. Bacterial growth is facilitated by peristalsis (which extensively mixes bacteria with fibers), and maintenance of optimal viscosity and temperature. However, the most astonishing feature promoting colonic bacterial growth is the virtual lack of mechanisms to suppress it.

It was initially believed that the immune system was tightly regulated to respond to pathogenic intestinal bacteria while tolerating commensal bacterial. However, it is increasingly recognized that bacterial residents of the large bowel cannot be easily divided as being pathogenic or nonpathogenic. Many of indigenous bacteria are known pathogens. As examples, Escherichia coli cause sepsis, Bacteroides cause abscesses, Enterococci cause endocarditis, and Clostridium histolyticum causes gas gangrene. These bacterial groups are considered normal inhabitants of the human colon because they can be found in every healthy person. Fluorescence in situ hybridization (FISH) analysis of the mucosal flora has clearly demonstrated that the host does not tolerate the indigenous microbiota, but separates them from contact with the mucosa.

Mucus barrier — Colonic and ileal biopsies from healthy persons are covered with mucus that is free of bacteria. The separation of fecal bacteria from the mucosa by mucus can be seen in sections of normal appendices, which were resected for suspected acute appendicitis and found to be normal (picture 3).

A similar separation of colonic bacteria from the mucosa can be observed in the distal colon of rodents. In contrast to humans, bacteria in the proximal colon of rats and mice directly contact the colonic wall and enter crypts in high concentrations [13]. However, contact of bacteria with the mucosa in the proximal colon of mice is selective. Long curly rods of E. rectale contact the mucosa and enter crypts in large numbers, while short coccoid rods of Bacteroides are separated from the colonic wall. The differences in arrangement of bacterial groups are especially obvious in multicolor FISH simultaneously visualizing different bacterial species.

Analysis of bacterial groups from the proximal colon of mice reveals that only long rods with a curly form contact the mucosa (E. rectale, Bifidobacteriaceae Lactobacillus). Short rods and coccoid bacteria such as Bacteroides, Enterobacteriaceae, Clostridioides difficile, Veillonella, and other groups are separated from the mucosa. The difference in the spatial distribution of differently shaped bacteria in the proximal colon of mice indicates that the mucus layer in the proximal colon of rodents is also present. However, because of a lower viscosity, it is penetrable for long corkscrew formed bacteria but not for short coccoid rods.

The shape of bacteria is important for their movement. Short rods are equipped with multiple pili that permit movements in a watery environment, but not in slime. Short rods have flagella, which act like propellers to move them through slime. Long, curly rods use complex body movements to screw through gels of high viscosity, but are immobile in water. Studies on velocity of differently shaped bacteria in simulated mucus with variable viscosity indicate that the coccoid Bacteroides bacteria have the highest velocity at viscosity corresponding to 0.2 percent agarose and are immobilized at 0.4 percent agarose, while the long, curly rods of the E. rectale group have the highest velocity at viscosity of 0.5 percent agarose. All bacterial movements stop at viscosity of 0.7 percent agarose in the simulated mucus [14].

The distribution of long, curly bacteria or short, coccoid rods within mucus can be used to mark the areas of changing viscosity. In healthy humans, the separation of bacteria from the mucosa is equal in the proximal and distal colon and bacteria are never found in crypts. In healthy rodents, the viscosity of the mucus in the proximal colon is markedly lower than in the distal colon, allowing bacteria with a long, curly rod shape to reach and contact the mucosa.

The reason for differences in mucus layer viscosity of the proximal colon in humans is unclear. The adherence of selected bacterial groups may have some unknown evolutionary advantages. The overall surface of a mouse colon is much smaller than that of a human. The reduced viscosity in the proximal colon creates compartments between crypts that contain bacterial stocks needed for restarting the colonic bioreactor.

The mucus layer is continuously renewed. Changes in viscosity of the mucus layer are controlled by two opposite processes. Mucus is solidified near the mucosa where active resorption of water takes place while it is diluted (and therefore becomes less viscous) near the lumen because of contact with water contained in the fecal stream. The viscosity of the mucus secreted by goblet cells is significantly lower than the viscosity of the dehydrated mucus film, which is attached to the columnar epithelium. The secreted mucus cannot combine with the dehydrated mucus because of differences in consistency. Instead, it pushes the dehydrated mucus toward the lumen and spreads below it. This process protects freshly secreted mucus from bacterial penetration until it is in turn solidified by water resorption and can serve as impenetrable cover. This process is visible histologically as onion-like stratification of the mucus layer, which can be seen in alcian stain, or even better with FISH (picture 4).

Biostructure of fecal microbiota — The structure of fecal microbiota within the intact intestine cannot be directly studied in humans since intestinal specimens filled with feces cannot be readily obtained from healthy persons. However, the spatial organization of fecal microbiota can be evaluated from stool specimens in which the outer layer represents the luminal surface of the intestinal mucus layer. The spatial structure of fecal microbiota can be investigated on sections of punched out fecal cylinders, which are then fixated and embedded in paraffin [15,16].The technique is analogous to the examination of stratified geologic structures by studying core samples.

In healthy humans, the surface of formed stools is covered with a mucus layer similar to the mucus covering the mucosal surface of biopsies. Fecal microbiota in healthy humans can be divided into habitual bacterial groups present in all subjects and occasional bacteria, which are present only in subgroups, either diffusely or locally.

The term "habitual" as used in this context is not synonymous with "commensal". Commensal bacteria include certain bacterial groups such as Bifidobacteriaceae, Enterobacteriaceae, Clostridia, Lactobacilli, and Enterococci, which are often present, but not obligatory for the healthy human intestine, and whose presence, absence, or alteration in number do not reflect whether the colon is healthy or diseased.

Studies evaluating 86 different bacterial groups have demonstrated that E. rectale (Roseburia spp.), Bacteroides, and F. prausnitzii groups are habitual and each comprise 20 to 50 percent of the fecal flora and together at least 70 percent of all bacteria present in feces [15,16]. All other bacterial groups occur only in a subset of patients.

With regard to the mucus layer, bacteria can be divided in fecomucous (bacteria that tend to live within feces), mucophob (bacteria that tend to avoid mucus), and mucotroph (bacteria that tend to live within mucus). All habitual bacteria are fecomucous; their highest concentrations are within feces, although they also enter mucus. Their concentrations diminish with increasing distance from the fecal surface. The mucophob bacteria (such as the Bifidobacteriaceae group) avoid mucus. Mucotroph bacteria (such as Enterobacteriaceae and Verucomicriaceae) are located on the border between feces and mucus.

The biostructure and composition of bacterial groups varies among individuals. Within individuals, the fecal biostructure and composition are generally stable from day to day but change substantially when studies have been performed repeatedly for more than six months.

DISRUPTION OF THE MUCUS BARRIER — A prominent feature of intestinal inflammation is disruption of the mucus barrier (picture 5). Once this occurs, bacteria migrate toward the mucosa where they build dense layers adherent to epithelial cells resulting in cytopathologic effects. Bacteria are generally located at the bottom of the crypts [9]. They can also be seen in regions that have been mechanically damaged (such as biopsy sites); thus, reports of submucosal bacteria should be interpreted cautiously unless they demonstrate the intact mucosal surface surrounding the presumed infiltration [9].

Despite massive adhesion, the epithelial barrier generally protects against deeper bacterial invasion. It is uncommon to find bacteria within epithelial cells or within submucosal regions even in patients with severe intestinal inflammation.

Interestingly, the highest concentrations of mucosal bacteria are found not in the inflamed regions of the intestine, but rather in relatively non-inflamed regions, suggesting that disruption of the mucus barrier occurs before the onset of intestinal inflammation. In inflamed regions, the bacterial concentrations are reduced, while leukocytes appear in the mucus in large numbers often forming arrays in the outer regions of the mucus. Leukocytes within mucus are absent in biopsies from healthy persons. Despite the high concentrations of leukocytes in mucus, the bacteria eventually reach the intestinal wall, leading to development of ulcers, fissures, and crypt abscesses.

Disruption of the mucus barrier and bacterial adherence to the mucosa is not specific to inflammatory bowel disease (IBD). Bacterial concentrations of 10(9) bacteria/mL or higher can be found within mucus in nearly all patients with IBD, but also in patients with celiac disease and nearly one-half of patients with a variety of conditions including acute diarrhea, diverticulosis, colon cancer or polyps and approximately 40 percent of patients with irritable bowel syndrome (IBS) [9].

Approximately 90 percent of bacteria found in mucus of patients with these diseases are represented by only three groups: Bacteroides, E. rectale, and F. prausnitzii (picture 5). The mean density of the mucosal bacteria is significantly lower in non-IBD disease controls and the composition of the biofilm differs. Bacteria of the Bacteroides fragilis group are responsible for >50 percent of the biofilm mass in IBD. By contrast, bacteria that positively hybridize with the Erec (E. rectale) and Fprau (F. prausnitzii) probes account for >50 percent of the biofilm in patients with IBS, but account for <30 percent of the biofilm in IBD.

Despite these differences, there is variability among individuals making differentiation among Crohn disease, ulcerative colitis, irritable bowel syndrome, diverticulosis, or colonic cancer based solely upon the fluorescence in situ hybridization (FISH) analysis of the mucosal flora at present impossible. Even F. prausnitzii, which is completely depleted in the feces of patients with Crohn disease, cannot be used as a reliable marker for diagnosis of Crohn disease. It can be detected in the mucus of colonoscopic biopsies from more than 50 percent of patients with Crohn disease, but it is absent in most of the biopsies from healthy persons.

The situation is different in patients with self-limiting colitis or specific infections such as Serpulina, Fusobacterium necrophorum (nucleatum), or Whipple's disease. In such patients, disruption of the mucus barrier is associated with an increase in bacterial groups other than Fprau+Erec+Bac so that they constitute 10 to 70 percent of bacterial mass.

Unrecognized pathogens — Several theories have been proposed to explain the increasing incidence of autoimmune and allergic conditions since the 20th century. One of these, the hygiene hypothesis, argues that improved hygiene and a lack of exposure to microorganisms of various types in early childhood have led to a lack of immunologic tolerance to ubiquitous environmental antigens.

Multiple studies have provided epidemiologic evidence in support of a link between higher hygienic standards and increased incidence of IBD. However, no studies have specifically examined the hygiene hypothesis as it pertains to the composition of the intestinal flora. There are also no data that point to major differences in the number or diversity of the colonic flora in individuals living in urban versus rural areas.

An alternative hypothesis related to microbiological exposures may be that the increase in allergies and atopic conditions is not directly due to lack of exposure to certain microorganisms, but rather due to exposure to previously unrecognized pathogens. The ubiquitous availability of produce from around the world and the mobility of modern society have led to the profound and rapid exchange of bacteria worldwide. As a result, exposure to facultative pathogens has likely increased. Concurrently, there may have been a shift from the more clinically apparent pathogens (such as Cholera, Salmonella, Shigella, and Yersinia) to those whose deleterious effects are more difficult to recognize. The clinical significance of H. pylori, for example, was not appreciated until the late 20th century.

The list of enteric bacteria that are potentially capable of causing disease is growing. Examples include Serpulina, Fusobacteria, adhesive Enterobacteriaceae, and Gardnerella, although their clinical importance is still being studied. Common to some of these organisms is the ability to compromise the mucus barrier and provide niches for growth of other enteric bacteria that normally do not have access to the mucosa (picture 6) [17].

Substances that reduce the viscosity of the mucus barrier — Another potential explanation for the hygiene hypothesis is that the decrease in microbiologic exposures is not the root of the problem but rather correlates with the introduction of ingested detergents that disrupt the intestinal mucus barrier. We know astonishingly little about the effects of detergents on the intestinal mucosa and the mucus barrier, despite their longtime use. However, in vitro and in vivo evidence suggests substances that reduce the viscosity of mucus thereby potentially contribute to bacterial proliferation on the intestinal mucosa.

Detergents — The addition of detergents such as dextran sodium sulfate (DSS) to an in vitro model of stimulated mucus enables migration of bacteria through gels with a viscosity corresponding to agarose concentrations of 0.9 percent [14]. Bacteroides migration could be seen in concentrations of up to 0.6 percent and migration of the E. rectale group up to 0.9 percent. Without detergents, Bacteroides is immobilized at viscosity corresponding to agarose concentration of 0.4 percent and the migration of all bacterial groups stops at viscosity corresponding to an agarose concentrations of 0.7 percent.

Although the effects of detergents on the mucosal barrier in humans are unknown, in the mouse model, the addition of DSS to food induces acute colitis, which becomes chronic after repeated exposure to DSS [18]. The DSS induced inflammation in mice is restricted to the large intestine, where bacterial concentrations are high, and bypasses the small intestine, where bacterial concentrations are low. Antibiotics relieve the DSS-induced inflammation.

DSS is absent in the human food supply. However, traces of dishwashing detergents are ingested with our food. The "cleaning" effects of ingested home soaps on colonic mucus have never been investigated.

Emulsifiers — Emulsifiers are another group of substances that could potentially influence the mucus barrier and have been used increasingly by the food industry since the beginning of the 20th century [19]. Data on IL-10 gene-deficient mice support the potentially detrimental role of emulsifiers, such as 2 percent carboxymethyl cellulose (CMC) [20]. High bacterial concentrations were found within crypts of Lieberkuhn in the ileum of all CMC-treated IL-10 knockout mice. The finding grossly resembled those found in the ileum of patients with Crohn disease.

Other causes — Several other substances can potentially perturb the mucus barrier, including bile salts, glutens, cigarette smoking, stress, and defensins.

BIOSTRUCTURE OF FECAL MICROBIOTA IN HEALTH AND INFLAMMATORY BOWEL DISEASE — The colon is a highly efficient bioreactor that is vulnerable to malfunction in the setting of inflammation or other gastrointestinal disorders. Most studies of the intestinal microflora have been based on homogenized stools samples, making it impossible to evaluate special perturbations in the organization of the colonic bioreactor. Contemporary techniques described above are revealing new insights into how the bioreactor is structured and responds to disease [15,16].

Functionally, the colonic bioreactor can be divided into three zones:

A transparent outer mucus layer, which is constantly dehydrated by the columnar epithelium, and is therefore highly viscous, impenetrable to bacteria, and separates the colonic bioreactor from the mucosa.

A central fermenting zone in which bacteria and fibers are stirred and fermented.

A transitional resting zone between the mucus layer and central zone in which mucus is increasingly diluted by luminal fluids and can be penetrated by bacteria. The softened mucus stays attached to the colonic wall for a prolonged time. Bacteria enter these soft portions of the mucus in concentrations that are inversely proportionate to the growing viscosity gradient, and as a result, they become increasingly immobilized (picture 7). Trapped within the resting zone, bacteria are protected against purging events and are therefore available to repopulate the bioreactor after occasional cleanouts such as during periods of fasting, diarrhea, or even antibiotic treatment. Thus, bacteria within the resting zone represent germinal stocks of the colonic bioreactor.

The firm mucus can be seen easily on alcian stain. In healthy subjects, both the resting (germinal) zone and the luminal (fermenting) area cannot be distinguished from each other. When the colonic bioreactor functions properly, the composition and density of bacteria found in these regions are similar. By contrast, in various diseases, the microbial changes in these compartments are different and disease specific.

Nonspecific changes of the colonic microbial biostructure — Several nonspecific changes to the colonic microbial biostructure occur in a variety of disease states:

Diarrhea of any cause is associated with increased thickness of the mucus layer and growing incorporation of the unstructured mucus within the fecal mass (picture 8). Patients with ulcerative colitis are an exception; in such patients the mucus layer is depleted.

A common feature of several intestinal disorders is a suppression of bacterial growth and metabolism at the center of the bioreactor. However, as a general rule, the suppression is most apparent with habitual bacterial groups. Their concentrations in healthy persons are especially high and the distribution throughout the fecal cylinder is normally homogenous. Initially, only the fluorescence signals fade. This fading is apparent in the phenomenon of hybridization silence, because the number of bacteria stays constant from suppressed to unsuppressed regions and only hybridization signals of bacteria change (relative hybridization silence). With progression, however, the hybridization signals of single bacterial groups may disappear completely (absolute hybridization silence) and it is impossible to discriminate between suppression and physical elimination of bacteria [15].

The epicenter of suppression is located in the center of feces, and with an exception in Crohn disease, does not involve the superficial "germinal" zone of the fecal cylinder, where the fluorescence of bacteria and bacterial numbers remain high (picture 9). The production of the suppressive substance must take place in the small intestine or at least upstream from the colon [15].

Changes of the colonic microbial biostructure in IBD — Changes involving the germinal bacterial zone are characteristic for active inflammatory bowel disease and absent in most other disease control groups. The most prominent feature of ulcerative colitis is a replacement of the mucus layer by leukocytes. The leukocytes are located in the germinal zone, which they progressively destroy (picture 10). Active Crohn disease is characterized by complete depletion of F. prausnitzii from the central and germinal zones of feces. The reproducible detection of these two features in three consecutive fecal cylinders taken in two-weekly intervals allows the diagnosis of active Crohn disease and ulcerative colitis with a 79 and 80 percent sensitivity and 98 and 100 percent specificity [16].

SUMMARY AND RECOMMENDATIONS

The surface of the intestinal tract in healthy individuals is free of bacteria in all bowel segments. Adherence of bacteria to epithelial cells is thus a sign of infection. By contrast, the intestinal lumen is never sterile.

The occurrence, composition, and organization of intestinal microbiota in each gut segment depend upon whether suppression (mechanisms that suppress bacterial growth) or separation (mechanisms that create a physical barrier from the host such as the mucus layer) dominates. (See 'Evolution's role in the distribution of intestinal microbiota' above.)

It was initially believed that the immune system was tightly regulated to respond to pathogenic intestinal bacteria while tolerating commensal bacterial. However, it is increasingly recognized that bacterial residents of the large bowel cannot be easily divided as being pathogenic or nonpathogenic. Many indigenous bacteria are known pathogens. As examples, E. coli cause sepsis, Bacteroides cause abscesses, Enterococci cause endocarditis, and C. histolyticum causes gas gangrene. The control of pathogens and the regulation of the colonic bioreactor require proper function of the intestinal mucus layer. Before bacteria can adhere and invade the mucosa, they must first traverse the mucus layer. (See 'Disruption of the mucus barrier' above.)

The structure and composition of intestinal bacteria can be markedly affected in the presence of a variety of diseases states. For example, inflammatory bowel disease is a polymicrobial infection that is characterized by a sustained broken mucus barrier, subsequent bacterial migration towards the mucosa and proliferation of a complex bacterial biofilm on the epithelial surface with resulting invasive and cytopathologic effects. (See 'Biostructure of fecal microbiota in health and inflammatory bowel disease' above.)

The composition and structure of fecal microbiota changes as a consequence of the inflammatory response. Active Crohn disease and ulcerative colitis can be distinguished from each other and other disease controls based upon the biostructure of fecal cylinders. (See 'Biostructure of fecal microbiota' above.)

  1. Kuramitsu HK, He X, Lux R, et al. Interspecies interactions within oral microbial communities. Microbiol Mol Biol Rev 2007; 71:653.
  2. Amann RI, Ludwig W, Schleifer KH. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 1995; 59:143.
  3. Amann R, Fuchs BM. Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques. Nat Rev Microbiol 2008; 6:339.
  4. Greuter D, Loy A, Horn M, Rattei T. probeBase--an online resource for rRNA-targeted oligonucleotide probes and primers: new features 2016. Nucleic Acids Res 2016; 44:D586.
  5. Swidsinski A, Göktas O, Bessler C, et al. Spatial organisation of microbiota in quiescent adenoiditis and tonsillitis. J Clin Pathol 2007; 60:253.
  6. Kandulski A, Selgrad M, Malfertheiner P. Helicobacter pylori infection: a clinical overview. Dig Liver Dis 2008; 40:619.
  7. Swidsinski A, Schlien P, Pernthaler A, et al. Bacterial biofilm within diseased pancreatic and biliary tracts. Gut 2005; 54:388.
  8. Scheithauer BK, Wos-Oxley ML, Ferslev B, et al. Characterization of the complex bacterial communities colonizing biliary stents reveals a host-dependent diversity. ISME J 2009; 3:797.
  9. Swidsinski A, Weber J, Loening-Baucke V, et al. Spatial organization and composition of the mucosal flora in patients with inflammatory bowel disease. J Clin Microbiol 2005; 43:3380.
  10. Franks AH, Harmsen HJ, Raangs GC, et al. Variations of bacterial populations in human feces measured by fluorescent in situ hybridization with group-specific 16S rRNA-targeted oligonucleotide probes. Appl Environ Microbiol 1998; 64:3336.
  11. Harmsen HJ, Raangs GC, He T, et al. Extensive set of 16S rRNA-based probes for detection of bacteria in human feces. Appl Environ Microbiol 2002; 68:2982.
  12. Dethlefsen L, Huse S, Sogin ML, Relman DA. The pervasive effects of an antibiotic on the human gut microbiota, as revealed by deep 16S rRNA sequencing. PLoS Biol 2008; 6:e280.
  13. Swidsinski A, Loening-Baucke V, Lochs H, Hale LP. Spatial organization of bacterial flora in normal and inflamed intestine: a fluorescence in situ hybridization study in mice. World J Gastroenterol 2005; 11:1131.
  14. Swidsinski A, Sydora BC, Doerffel Y, et al. Viscosity gradient within the mucus layer determines the mucosal barrier function and the spatial organization of the intestinal microbiota. Inflamm Bowel Dis 2007; 13:963.
  15. Swidsinski A, Loening-Baucke V, Verstraelen H, et al. Biostructure of fecal microbiota in healthy subjects and patients with chronic idiopathic diarrhea. Gastroenterology 2008; 135:568.
  16. Swidsinski A, Loening-Baucke V, Vaneechoutte M, Doerffel Y. Active Crohn's disease and ulcerative colitis can be specifically diagnosed and monitored based on the biostructure of the fecal flora. Inflamm Bowel Dis 2008; 14:147.
  17. Swidsinski A, Mendling W, Loening-Baucke V, et al. Adherent biofilms in bacterial vaginosis. Obstet Gynecol 2005; 106:1013.
  18. Okayasu I, Hatakeyama S, Yamada M, et al. A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology 1990; 98:694.
  19. Chassaing B, Koren O, Goodrich JK, et al. Dietary emulsifiers impact the mouse gut microbiota promoting colitis and metabolic syndrome. Nature 2015; 519:92.
  20. Swidsinski A, Ung V, Sydora BC, et al. Bacterial overgrowth and inflammation of small intestine after carboxymethylcellulose ingestion in genetically susceptible mice. Inflamm Bowel Dis 2009; 15:359.
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