Introduction

The digestive tract is constantly interacting with the external environment, being the major immunologic organ of the body. It is responsible for immune tolerance as well as for immune activation in case of infections. Most of these functions are made possible by the continuous crosstalk between gastrointestinal mucosa and microorganisms contained in the intestine, commonly called gut microbiota. The gut microbiota, the whole intestinal flora of the gastrointestinal tract, is a complex microbiological system that is attracting increasing interest from the scientific community. It is estimated that the total number of bacteria in the human body is 10 to 100 trillion, at least 10 times more than the total number of somatic and germ cells of the human body, with bacterial genes in a ratio of 100:1 with human genes.

To date, studies of gut microbiota have been limited because of technical issues in the identification and study of microbial components. The number of species present in human intestine is between 500 and 800, of which 80% cannot be cultured in the laboratory with available techniques, but new methods are now available, at least for research purposes, and include denaturing gradient gel electrophoresis (DGGE) analysis and microarray, and these techniques are already changing the understanding about the physiology of the digestive system.

Mucosal barrier, gut microbiota, and intestinal health

Gut microbiota can be considered an organ in the human body, and, because of its continuous and constant crosstalk with intestinal mucosa, it supports several physiologic processes including the intestinal barrier effect, immune system modulation (inducing tolerance and/or activation), metabolic and trophic functions through production of butyric acid or other products, drug and toxin metabolism, and behavior conditioning. These functions are possible because of its direct or indirect interaction with components of the intestinal mucosa. Direct interaction occurs with enterocytes, dendritic cells and intraepithelial immune cells. Indirect interaction occurs for immune cells and nonimmune cells or the noncell population of the intestinal mucosa. Immune cells within the lamina propria are mononuclear cells, lymphocytes, polymorphonuclear cells, plasma cells, dendritic cells, and macrophages. Immune cells are organized, particularly in the small intestine, in microstructures, called Peyer patches, and in lymphoid tissue associated with gastrointestinal mucosa. Nonimmune cells, whose role in intestinal homeostasis is also important, are mesenchymal cells, endothelial cells, fibroblasts, and muscular and nervous cells.

Under physiologic conditions, there is a continuous recirculation of immune cells from peripheral blood to lymph nodes: this balance is defined as physiologic inflammation caused by antigens of the normal intestinal flora and diet.

The recognition of antigens depends on specific or nonspecific mechanisms. Nonspecific mechanisms are responsible, for instance, for the recognition of lipopolysaccharides or peptidoglycans, and are shared by diverse cellular subtypes responsible for innate immunity. Toll-like receptors (TLRs) and NOD-like receptors (NLRs), the most important innate immunity pathways, are the major armamentarium for both immune and nonimmune cells to sense gut microbiota. In contrast, specific antigens are recognized by immune cells through mechanisms mediated by human leukocyte antigen.

Under pathologic chronic conditions such as in chronic diarrhea, infections, irritable bowel syndrome (IBS), or inflammatory bowel disease (IBD), defects in the intestinal barrier are thought to be the reason why gut microbiota or its components interact not just with enterocytes, dendritic cells, or intraepithelial immune cells but also with other cellular components of intestinal mucosa, so that physiologic inflammation becomes a pathologic inflammation, with the activation of all intestinal cell components.

Mucosal barrier, gut microbiota, and intestinal health

Gut microbiota can be considered an organ in the human body, and, because of its continuous and constant crosstalk with intestinal mucosa, it supports several physiologic processes including the intestinal barrier effect, immune system modulation (inducing tolerance and/or activation), metabolic and trophic functions through production of butyric acid or other products, drug and toxin metabolism, and behavior conditioning. These functions are possible because of its direct or indirect interaction with components of the intestinal mucosa. Direct interaction occurs with enterocytes, dendritic cells and intraepithelial immune cells. Indirect interaction occurs for immune cells and nonimmune cells or the noncell population of the intestinal mucosa. Immune cells within the lamina propria are mononuclear cells, lymphocytes, polymorphonuclear cells, plasma cells, dendritic cells, and macrophages. Immune cells are organized, particularly in the small intestine, in microstructures, called Peyer patches, and in lymphoid tissue associated with gastrointestinal mucosa. Nonimmune cells, whose role in intestinal homeostasis is also important, are mesenchymal cells, endothelial cells, fibroblasts, and muscular and nervous cells.

Under physiologic conditions, there is a continuous recirculation of immune cells from peripheral blood to lymph nodes: this balance is defined as physiologic inflammation caused by antigens of the normal intestinal flora and diet.

The recognition of antigens depends on specific or nonspecific mechanisms. Nonspecific mechanisms are responsible, for instance, for the recognition of lipopolysaccharides or peptidoglycans, and are shared by diverse cellular subtypes responsible for innate immunity. Toll-like receptors (TLRs) and NOD-like receptors (NLRs), the most important innate immunity pathways, are the major armamentarium for both immune and nonimmune cells to sense gut microbiota. In contrast, specific antigens are recognized by immune cells through mechanisms mediated by human leukocyte antigen.

Under pathologic chronic conditions such as in chronic diarrhea, infections, irritable bowel syndrome (IBS), or inflammatory bowel disease (IBD), defects in the intestinal barrier are thought to be the reason why gut microbiota or its components interact not just with enterocytes, dendritic cells, or intraepithelial immune cells but also with other cellular components of intestinal mucosa, so that physiologic inflammation becomes a pathologic inflammation, with the activation of all intestinal cell components.

Chronic diarrhea and gut microbiota: when intestinal homeostasis is disturbed

Chronic diarrhea is characterized by the presence of diarrhea for more than 4 weeks, and it requires a different diagnostic and therapeutic work-up than acute diarrhea. In developing countries, chronic diarrhea is frequently caused by chronic bacterial, mycobacterial, and parasitic infections, although functional disorders, malabsorption, and IBD are also common. In developed countries, common causes of chronic diarrhea are IBS, IBD, celiac disease, malabsorption syndromes, including lactose intolerance and small intestinal bacterial overgrowth (SIBO), or chronic infections, particularly in immune-compromised patients. All these conditions have been associated directly or indirectly with gut microbiota alterations (or dysbiosis). In order to classify the involvement of gut microbiota in chronic diarrheal disorders, 3 major groups of alterations are recognized:

• 1.
  

  Gut microbiota alteration in the presence of infectious agents

  
  
• 2.
  

  Gut microbiota alteration in the presence of intestinal inflammation and damage

  
  
• 3.
  

  Gut microbiota alteration as the major determinant of disease without signs of intestinal inflammation or damage

In the first group, main determinants are represented by pathogens, whose mechanisms of damage have been extensively studied. It is well known that many bacterial species act through the production of toxins, including Escherichia coli , Shigella dysenteriae (with the so-called Shiga toxin); Clostridium perfringens ( C perfringens enterotoxin, a-toxin, b-toxin, and u-toxin); Clostridium difficile (toxins A and B), Staphylococcus aureus (a-hemolysin), Bacillus cereus (cytotoxin K and hemolysin BL), and Aeromonas hydrophila (aerolysin, heat-labile cytotoxins, and heat-stable cytotoxins). These toxins can cause diarrhea by different mechanisms, including direct action of toxins on intestinal epithelial ion channels; indirect interaction with ion transporters; and cytotoxic, hemolytic, proinflammatory action resulting in intestinal mucosal integrity loss with reduction of normal absorptive capacity. Other studies highlight how bacterial toxins or other related substances interact with other cell types deeper within the intestinal mucosa, including neuronal cells and other mesenchymal cells, modifying their functions. It is possible that pathogens can determine alterations within other populations of gut microbiota, sustaining paraphysiologic changes of intestinal homeostasis.

The second group of alterations includes chronic disorders with multifactorial pathogenesis, such as IBD, for which it remains unclear whether gut microbiota alterations are the cause or the consequence of the disease. Both qualitative and quantitative changes of the microbial population have been described, together with an increased prevalence in selective microbial strains. Crohn disease and ulcerative colitis (UC) frequently affect intestinal areas with the highest microbial concentration, such as the terminal ileum and colon, where bacterial metabolism and fermentation processes are more intensive. Darfeuille-Michaud and colleagues showed a significantly increased concentration of enteroinvasive E coli in the terminal ileum of patients with Crohn disease, and an increased concentration of tumor necrosis factor (TNF)-α produced by macrophage activation in response to these bacterial strains. Rodemann and colleagues reported an increased number of infections by C difficile , increasing from 4% in 2003 to 16% in 2006, whereas Swidsinsky and colleagues showed that the concentration of mucosal bacteria, particularly those adhering to the intestinal mucus and enterocytes, were higher in patients with IBD (particularly Crohn disease) compared with healthy subjects. In patients with Crohn disease, higher concentrations of anaerobes, especially Bacteroides , has been found in the ileum and in other segments of the colon during active disease. Frank and colleagues showed a decrease in bacterial biodiversity in IBD gut microbiota with a low prevalence of Firmicutes methane-producing bacteria, an increase in Enterobacteriaceae and hydrogen-producing bacteria, and a reduction of short-chain fatty acids and epithelial trophism. Similar results were reported by Scanlan and colleagues in 2008.

An additional group of alterations accounts for diseases without major signs of intestinal inflammation or damage, like IBS. Up to 84% of patients with IBS have an abnormal lactulose hydrogen breath test (LBT), suggesting the presence of SIBO. Moreover, symptoms associated with IBS (bloating, abdominal pain, and altered bowel habits) are generally similar to those associated with SIBO. Studies based on new molecular biology techniques have highlighted significant differences in the quality, quantity, and temporal stability of gut microbiota in patients with IBS compared with healthy controls. In particular, fecal samples from patients with IBS have lower levels of coliform bacteria, Lactobacilli , and Bifidobacterium species, and higher numbers of Clostridium species and Enterobacteriaceae compared with controls. Patients with diarrhea-predominant IBS had a decreased number of Lactobacillus species, whereas constipated patients with IBS showed an increase in the number of Veillonella species compared with healthy controls. Gut microbiota from patients with IBS were less stable over time compared with healthy subjects, as shown by DGGE fingerprint profiles ; the composition of mucosa-adherent microbiota is different between patients with IBS and healthy controls, with Eubacterium rectale – Clostridium coccoides being the predominant bacterial group, accounting for 48% of the total adherent bacteria in IBS compared with 32% of healthy controls. In addition, the density of the bacterial biofilm (a layer of microorganisms that forms a coat on the surface of the intestine) was significantly larger in patients with IBS. More recent data have reinforced these observations, showing that microbiota from patients with IBS and healthy controls also differed in other bacterial species including Coprococcus spp, Collinsella spp and Coprobacillus spp. It can be assumed that the increasing availability of new microbiological techniques will result in increasing knowledge that may lead to new therapeutic approaches.

Studying gut microbiota

The major limitation of current microbiologic methods is that the classic culture-dependent techniques can detect less than 30% of bacterial species. Culturing the jejunal aspirate identifying and counting colony-forming units (CFU) is currently the gold standard in diagnosing SIBO. This method, which does not characterize the gut microbiota responsible for SIBO, is invasive; for this reason other techniques are used that are based on measuring functional alterations caused by gut microbiota. These approaches include glucose breath tests (GBTs) and LBTs, which are currently widely used in clinical practice. Among them, GBT has a higher diagnostic accuracy in studies comparing breath tests with cultures. Certain conditions, such as hypochlorhydria, anatomic abnormalities, or gastrointestinal motility failure, may cause SIBO and consequently malabsorption. In these cases, GBT may be useful to establish whether malabsorption is caused by SIBO. Data regarding the role of SIBO in IBS are still inconclusive, and routine screening for SIBO is not recommended. GBT and LBT are hydrogen (H ₂ )-based breath tests, in which the determination of hydrogen (and methane) concentration in expired air is used to assess bacterial carbohydrate metabolism. Hydrogen (and methane) is normally produced in the colon by the fermentation of residual carbohydrates by gut microbiota. In SIBO, glucose, lactulose, or other sugars (including lactose or fructose) can be fermented by bacteria in the small bowel. Based on different studies and on a recent consensus conference, GBT is the breath test with the best diagnostic accuracy for diagnosing SIBO.

Besides being accurate and noninvasive, H ₂ -breath tests have other advantages, such as lack of toxicity, low cost of substrates, and easy accessibility in clinical practice.

For the study of gut microbiota, novel and culture-independent techniques have become available that are based on the analysis of bacterial 16S rRNA or ribosomal RNA. These methods have revealed the myriad of bacterial lineages within the human gut. These techniques use quantitative polymerase chain reaction, DGGE, DNA sequencing, microbial gene microarray analysis, and fluorescence in situ hybridization. Major limitations of these approaches are the costs and the need for expert technologists who are able to extract, handle, and process the fecal or intestinal biopsy samples, and to interpret results. Despite intriguing experimental data, a full characterization of gut microbiota from a healthy subject is not yet possible. Furthermore, the reproducibility of results has not been studied between different centers. In addition, it is not clear whether characterization of gut microbiota should be performed on feces or on intestinal biopsies and which conditions (ie, diet, time of collection, number of samples) should be observed. Further studies are needed to provide a more robust and oriented basis for future interventional studies of microbiota modulation as a therapeutic approach for gastrointestinal and extraintestinal diseases.

Gut microbiota modulation

Gut microflora stability is influenced by mechanisms that directly affect gut microbiota composition as well as by mechanisms dependent on host physiology.

Gut microbiota modulation can be achieved by:

• 1.
  

  Antibiotics and agents directly affecting vitality of germs

  
  
• 2.
  

  Probiotics: single or multiple specific bacterial or yeast species that reach the intestinal microbiota and interact with it and with gut mucosa, thereby resulting in transient gut microbiota alteration and clinical benefit to the host

  
  
• 3.
  

  Prebiotics: substrates able to promote growth and metabolism of certain strains, thereby resulting in a transient alteration or stabilization of the gut microbiota

Factors of host physiology that determine gut microbiota include:

• 1.
  

  Gastric acid secretion

  
  
• 2.
  

  Biliary, pancreatic, and intestinal secretion

  
  
• 3.
  

  Visceral motility

  
  
• 4.
  

  Mucosal structural integrity

Alteration of any of these mechanisms can predispose to gut microbiota alterations (or dysbiosis). For this reason, many factors interacting with these mechanisms can modify gut microbiota homeostasis.

For example, the widespread use of proton pump inhibitors (PPIs) has been associated with an increase of enteric infections: the hypochlorhydria induced by PPIs increases the translocation of Salmonella typhimurium , Shigella flexneri , Campylobacter jejuni , and enterotoxic E coli in enteric mucosa. PPIs increase the infection rate of C difficile and predispose to SIBO. Motility alterations also can predispose to intestinal dysbiosis: in Parkinson disease there is an alteration of intestinal motility that correlates with a high rate of SIBO. In diabetes, intestinal motility may be disturbed, thereby altering microbial composition and mucosal integrity and predisposing to bacterial overgrowth and mucosal inflammation.

Different approaches based on the control of these 4 factors can modulate gut microbiota composition. The use of prokinetics, for example, can increase intestinal motility and reduce luminal fermentations and bacterial growth ; diet low in carbohydrates can reduce insulin resistance and obesity, and modulate the composition of gut microbiota; the reduction of PPI use can decrease the number of pathogenic bacteria on the intestinal mucosa.

Later in this article, the focus is on agents used for modulation of gut microbiota.