The need to shed light on the unknown aspects of pathophysiology of common disorders, such as gastrointestinal ones, has led researchers through last decades to study and define the role of microorganisms within the human intestine and their interactions with the host. The progress of technology has permitted the overcoming of culture-based methods to study microbes and paved the way to molecular techniques, which allow the analysis of microbial genome, microbial functions, and metabolism. These progresses opened a window on the world of microbiology and permitted to deepen into the key role played by gut microbiota and dysbiosis in health status and diseases, both gastrointestinal and extraintestinal. So, scientists focused their attention in developing new strategies to restore eubiosis and to manipulate gut microbes by modifying dietary habits, administrating antibiotics, probiotics, and prebiotics and using fecal microbiota transplantation as treatment of gastrointestinal, infectious, cardiovascular, metabolic, immune-mediated, neuro-psychiatric, and oncological disorders. The next challenges will be to elaborate standard protocols with definite outcomes predictors in disease-specific settings.

Gastrointestinal disorders have always had a very high prevalence in the world population and still today people frequently ask for a gastroenterological consultation because of them [1, 2]. As many other common disorders, they often tend to become chronic, symptoms relapse despite therapies, and the patients’ quality of life and work productivity are significantly impaired [3-5]. The most usual are functional disorders, like irritable bowel syndrome (IBS) and functional dyspepsia, though the last decades have seen a rise of inflammatory bowel diseases (IBD), characterized by chronic inflammation of the intestine [5-7]. The burden of health care management related to these conditions is considerable as, in most cases, treatments contemplate a multidisciplinary approach, not only due to the complexity of the therapies but also because of the need to support the patients’ global health, including consultations from nutritionists, psychologists and psychiatrists, surgeons, and other specialists [2, 8, 9].

The knowledge of the pathophysiology of these diseases has increased through the years and it is still growing, and we are aware that they are due to multifactorial conditions; however, many sides of these mechanisms remain unclear even nowadays. Moreover, in most instances, current therapies only partially resolve symptoms and prevent disease relapse and progression, and we are far from identifying a definitive cure that targets the pathophysiological processes [10-13]. These pending issues led researchers and physicians to focus their attention through the ages on the role played by microbes within the host and, consequently, to study human interactions with microorganisms. Indeed, it was noted that both IBS and IBD may be triggered or exacerbated by an episode of infective acute gastroenteritis, which may cause persistent subclinical inflammation, altered gut motility and alteration of intestinal barrier function [14, 15]. Moreover, interventions aimed at modulating intestinal microbiota (diet, antibiotics, probiotics, prebiotics, and fecal microbiota transplantation) have been associated to an improvement of gastrointestinal symptoms in these conditions, even though with some still unclear aspects [16, 17].

Nowadays, thanks to the new technologies, which permit an extensive gut microbiota profiling, we can claim that, due to the vastity of roles played by intestinal microbes, including those regarding chemical transformations of nutrients, inflammatory and immunoregulatory effects, barrier and metabolic functions, human gut microbiota can be effectively considered as a real organ, whose equilibrium perturbances may set up disease promoting conditions [18]. Indeed, as microbes existed for billions of years before humans [19], it is logical to think that human beings coevolved with them symbiotically, creating a superorganism [20]. Over the years, it was revealed that gut microbiota homeostasis is crucial not only for gastrointestinal and global wellness but it is also involved in immune-mediated diseases, metabolic and cardiovascular disorders, oncological conditions, and neuro-psychiatric diseases [21].

Time has passed since the seventeenth century, when the Dutch scientist Antony van Leeuwenhoek discovered “animalcules” and other microorganisms by the use of a microscope and started the revolution of microbiology [22]. Then, in the first decade of the twentieth century, scientists began to use tissue samples for isolation of viruses [23] and, subsequently, bacterial cultures to identify single infective pathogens for the study of virulence and antibiotic susceptibility became available [24].

Lactulose and glucose breath tests spread in the second half of the 1,900 to diagnose small intestinal bacterial overgrowth and orocecal transit [25]. Until modern times, researchers were focused mainly on pathogenic microbes and the interest in studying nonpathogenic colonizing microorganisms came out when the evidences of their impact on human health and disease became evident [26].

The need of culture-independent techniques came up when the scientific community accumulated sufficient evidence to recognize the importance of studying commensal flora, as culture-dependent tests were limited by the impossibilities and difficulties of identifying some species, as in the case of obligate anaerobes [27]. In the 1990s, these necessities were solved by molecular techniques, as the amplicon sequencing of the 16S ribosomal RNA, a 1,500 nucleotide long sequences from small ribosomal subunits used as a genetic marker of diversity [28]. To identify the different bacterial species, this method studies the differences of nucleotides in the V1–V9 hypervariable regions by linking specific primers [27]. According to the similarities of the sequences, these are clustered into operational taxonomic units, which are related to the phylogenesis of the species. A more extensive approach is shotgun metagenomics sequencing, which allows to sample the whole genome of a microbial community by using random primers [29]. This method is more expensive and need more informatic resources, but it permits to elucidate the whole taxonomic composition of strains and the functions of a microbial system [30].

The introduction of next-generation sequencing in the 2000s, which allows simultaneous sequencing of a large number of small fragments of DNA, has led to an abatement of costs and to a significant reduction of sequencing time, and permitted the study of diverse microbiomes in their native environments [31, 32]. The Human Microbiome Project, a worldwide program, which has the main aim to characterize the whole human microbiome between different communities and its influence on physiology and disease, has made wide use of these techniques [33].

The progress of technology allowed to integrate the metagenomic analyses with the study of genes expression of bacterial communities by sequencing mRNA molecules, namely, metatranscriptomics [34]. This strategy can reveal details of transcriptionally active bacterial species, obtaining information about the functional status of gut microbes. Another step ahead in understanding microbial functions and metabolism was made with the introduction of metaproteomics, which uses spectrometry to assess the whole amount of proteins in a certain sample [35], and metabolomics, that studies and quantifies the metabolome of a biological system, consisting of small molecules involved in chemical processes [36].

Together, the combination of “omic” techniques allows to admire the comprehensive panorama of composition and functions of both fecal and mucosal gut microbiota, and its interaction with the host [37].

Through the last decades, the knowledge about the complex interactions between microbes, human biology, and pathophysiology went far beyond the simple relation “one pathogen-one disease.” Considering the example of Helicobacter pylori, a renowned Gram-negative bacterium which is associated not only to peptic ulcer disease but also to gastric adenocarcinoma, gastric lymphoma, colon cancer and adenomatous polyps, sideropenic anemia, and immune thrombocytopenic purpura, it is easy to imagine how gut microbes may be involved in the development of many intestinal and systemic diseases [38, 39].

The microbiota harbored in humans is composed by viruses, fungi, protozoa, archaea, and mainly bacteria, which are estimated to be approximately 1013 cells in the whole body with a ratio of bacterial to human cells of 1:1 [40]. Bacterial micro-organisms are most represented in the gastrointestinal tract and mainly belong to the phyla Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, and Verrucomicrobia [41]. Dysbiosis is the loss of balance of microbial population, which implies a decrease in overall diversity with reduction of beneficial microbes and increase of pathobionts; it is caused by perturbing factors (such as diet, stressful events, infections, or antibiotics), and it is involved the developing of many diseases [42].

Primarily, a decrease in biodiversity and an increase of Proteobacteria, mainly Escherichia coli, have been widely associated with IBD [43, 44]. A reduction of the butyrate producing phyla Firmicutes and Bacteroidetes and a decrease of the ratio Bacteroidetes: Firmicutes was reported as well [45, 46]. Also Fusobacterium spp. has been isolated more frequently from tissue samples of IBD patients than healthy controls [47]. In treatment-naïve children with new onset of Crohn’s disease, an increase of Enterobacteriaceae, Pasteurellacaea, Veillonellaceae, and Fusobacteriaceae and a reduction of Erysipelotrichales, Bacteroidales, and Clostridiales were reported on gastrointestinal tissue samples [48]. Ulcerative colitis (UC) patients show a decrease of Akkermansia muciniphila, Butyricicoccus pullicaecorum, Clostridium colinum, and Roseburia [49]. However, whether dysbiosis is a cause or a consequence of bowel inflammation, it still remains uncertain [43]. It is broadly accepted that IBDs arise from an intense inflammatory immune-mediated response to gut microbes in genetically predisposed individuals [50].

In general, patients with IBS have a reduced gut microbial diversity; furthermore, many studies show a common shift of microbiota composition, often distinguished by an increase of Proteobacteria, (especially Enterobacteriaceae) and Firmicutes, including Lactobacillus and Ruminococcus genera, frequently associated to a reduction of Actinobacteria (mainly bifidobacteria), Faecalibacterium, and methanogenic bacteria [51, 52].

As certain gut microbes have already been associated to specific cancers due to chronic inflammation mechanisms or genotoxicity, like in the cases of H. pylori in gastric cancer or Salmonella typhi in biliary cancer, the hypothesis of carcinogenetic mechanisms linked to dysbiosis has been postulated [53]. Indeed, there is an intensive bidirectional crosstalk between the gut microbiota and the host’s nervous system through hormones and active molecules that can affect tumorigenesis by the production of anticancer molecules or by triggering immune-mediated responses against tumor development, or, on the contrary, by promoting cancer with similar pathways [54]. As an example, Fusobacterium nucleatum, which has been associated with colon adenocarcinoma, may inhibit Natural Killer cells cytotoxicity and the activity of tumor infiltrating lymphocytes [55]. Moreover, E. coli strains producing the genotoxic compound colibactin, which has been identified in colorectal cancer biopsies, may enhance tumor growth by producing growth factors in preclinical models [56].

Dysbiosis has been widely associated to many autoimmune conditions, and the role of gut microbes in inducing the loss of immune tolerance in individuals with genetic susceptibility seems a key factor [57]. The finding of anti-Saccharomyces cerevisiae antibodies in antiphospholipid syndrome, systemic lupus erythematosus (SLE), and rheumatoid arthritis it’s a clue; indeed, they were found to have an overlapping structure with common autoantigens [58]. Several studies reported microbial imbalances in different immune-mediated pathologies: patients with early rheumatoid arthritis show a significant reduction of bifidobacteria, Bacteroides, Porphyromonas, and Prevotella if compared to individuals suffering from fibromyalgia [59]; there is an increase of the relative abundance of Proteobacteria in SLE patients [60]; and patients with SLE or Sjögren’s syndrome have a reduced bacterial richness, low Firmicutes:Bacteroidetes ratio, and an increase of Bacteroides in stools [61]; an imbalance in microbial diversity toward bifidobacteria, Bacteroides, and E. coli during young age may influence immune modulation and trigger susceptibility to type 1 diabetes mellitus [62].

Several clinical studies reported alterations of microbial population in metabolic syndrome and type 2 diabetes mellitus. For instance, it is described that Firmicutes are increased in lean compared to overweight males [63]. Besides, diabetic patients are characterized by a reduction of butyrate-producing bacteria (such as Faecalibacterium prausnitzii and Roseburia intestinalis), together with an increase of E. coli, Eggerthella lenta, and clostridia [64]. A reduction of microbial diversity has been associated with an increase of insulin resistance, inflammation, and adiposity.

Of interest, also cardiovascular diseases seem to have a relevant pathogenic substrate in gut microbial composition. Enterobacteriaceae and Streptococcus spp. are more represented in individuals with atherosclerosis [65], and the genus Collinsella is reported to be increased in symptomatic atherosclerosis cases [66]. Trimethylamine N-oxide, a product of microbial metabolism, has a role in atherogenesis [67], and it has been assessed that blood levels of this compound in acute coronary syndromes are a predictor of adverse cardiac events [68].

As it is known that a connection between gut microbiota and brain exists and that intestinal microbes are crucial in neurodevelopment and neurodegeneration, it is not surprising that neurological and psychiatric disorders have been linked to disturbances of microbial normal composition [69]. As an example, it has been reported that Prevotellaceae in stools of patients with Parkinson’s disease are reduced and that the abundance of Enterobacteriaceae correlated with the grade of motor disturbances [70]. Moreover, an increase of genera Clostridium spp., Desulfovibrio spp., and, Lactobacillus spp., a reduction of Bifidobacterium spp. and Prevotella spp., together with a decrease of the Bacteroidetes:Firmicutes ratio has been assessed in individuals with autism specter disorders [71-73]. Preclinical evidence shows that gut bacteria transplantation from depressed humans induces depressive symptoms in germ-free mice, but currently data on humans about a precise association between major depression and specific microbial imbalance are still inconsistent [74].

Various factors may influence gut microbial composition, and the restoration of eubiosis through different strategies is supposed to be the key of resolution or amelioration of many diseases [75]. Considering the different abundance of gut microbes in different clinical conditions, specific modulations of the reduced or increased species may allow to manipulate the state of illness.

As previously mentioned, dietary habits are essential for the regulation of intestinal microbial populations, and changing our nutrition may exert microbiota-induced beneficial effects. For instance, a higher bacterial diversity with an increase of butyrate-producing microbes has been associated with Mediterranean diet, consumption of fat sources derived from vegetables and poly- and monounsaturated fats, which are generally linked to a healthy status [76]. A diet with low intake of fermentable oligo-, di-, mono-saccharides, and polyols, whose assumption can cause water retention and fermentation by gut microbes with hydrogen or methane production and distension of bowels, is reported to mitigate symptoms in patients with IBS, even if with a reduction of bifidobacteria and total bacterial abundance after a short-term fermentable oligo-, di-, mono-saccharides, and polyols restriction [77]. A study in a mouse model of pancreatic cancer assessed a decline of tumor growth and a change in gut microbial composition, with a reduction of the proinflammatory microbes (Bacteroides acidifaciens, E. coli, and others) and an increase of the anti-inflammatory butyrate-producing bacteria (mainly Lachnospiraceae), after a fiber-rich food regimen with resistant starch [78]. Another recent preclinical trial demonstrated that fasting-mimicking diet and refeeding in a mouse model of chronic colitis can improve IBD-associated symptoms and histology through modulating gut microbiota and other mechanisms [79]. In a near future, new evidences may pave the way for engineered diets with selected beneficial components and restriction of detrimental aliments to modulate gut microbiota to treat or prevent diseases, included IBD and tumors [80].

The most intuitive way to modulate gut microbes is through antibiotics, which are not only imputable of causing dysbiosis. The clearest example is rifaximin, a low toxicity rifamycin derivative poorly absorbable antibiotic with strong antibacterial effect both on aerobic and anaerobic gram-positive and gram-negative bacteria [81]. Despite its antibiotic properties, rifaximin has also an eubiotic function: indeed, it can promote the growth of favorable bacteria, such as bifidobacteria and lactobacilli, without altering microbial diversity [82]. Moreover, rifaximin seems to have an anti-inflammatory activity, as assessed by the reduction in interleukin-8 and matrix metalloproteinase-9, and its cyclic use is of benefit in treating of symptomatic uncomplicated diverticular disease [83]. This drug is also efficient in preventing cirrhosis complications, such as spontaneous bacterial peritonitis and hepatic encephalopathy recurrence, and its use is associated to reduced endotoxemia and to a functional change of microbial metabolic profile [84]. Several studies reported the effectiveness of rifaximin in patients with IBS and small intestinal bacterial overgrowth compared to placebo [85]. In IBD, there is evidence of effective systemic antibiotic use in disease complications, such as septic ones and fistulas or in chronic pouchitis, but data are still lacking about microbiota-targeted therapy in different phase of activity, which may have a complementary role in future management [86]. Recent findings indicate that antibiotics may be a possible future treatment also in autoimmune diseases, as preclinical models show that broad-spectrum antibiotics can increase T-regs in the intestinal lamina propria and in extraintestinal lymphoid tissues, decrease effector T cells and inflammatory cytokines together with microbial diversity alterations [87].

Administration of probiotics, which are “live microorganisms that when administered in adequate amounts confer a health benefit on the host,” [88] has been studied as prevention strategy or treatment of different diseases. For instances, Lactobacillus rhamnosus GG and Saccharomyces boulardii may be beneficial in preventing diarrhea caused by dysbiosis associated to antibiotic therapies and Clostridium difficile infection [89, 90]. The use of probiotics has been applied in the field of IBD too; indeed, E. coli Nissle 1917 and VSL#3 (a mixture of 8 probiotics strains) are frequently prescribed in clinical practice to manage mild-to-moderately active disease flare, mostly in UC [91, 92]. Some authors reported an improvement in specific scores in anxious patients by administering probiotics (mainly lactobacilli), but studies used different measurements methods, strains, dosages, and treatment periods with lack of standardization [93]. A systematic review evaluated 13 randomized controlled trials about probiotic administration compared to placebo in preventing acute upper respiratory tract infections and assessed a significant reduction of the infection rate, of the infectious episode length, of the necessity to use antibiotics, and of the cold-related school absence, even with a low quality of evidence [94]. Supplementation with lactobacilli seems also effective in preclinical colorectal cancer model to prevent precursor lesions, tumor onset, and growth [95]. Many other studies have been conducted in various clinical conditions, such as metabolic disease, IBS, chemotherapy-induced diarrhea, urinary tract infections, and liver encephalopathy, but evidence is poor [96], and understanding if the efficacy of probiotics is strain-specific and disease-specific is still a major challenge in clinical trials [90].

Gut microbial modulation is also possible by administrating prebiotics, which are defined as nondigestible compounds that, through their metabolization by microorganisms in the gut, modulate composition and/or activity of the gut microbiota, thus conferring a beneficial physiologic effect on the host [97]. These compounds are mainly from subset of carbohydrate groups and include fructans, galacto-oligosaccharides, lactulose, starch, and glucose-derived oligosaccharides, as well as noncarbohydrate oligosaccharides [98, 99], which can be fermented by gut bacteria and enhance production of short-chain fatty acids, immunomodulation, and improvement of barrier functions [100]. They might be used alone or in synergic association with probiotics, as synbiotics [101]. Prebiotics may be of benefit in acute gastroenteritis via contrasting pathogens by blocking their adhesion to bowel mucosa, displacing them, or reducing their virulence [102]. Currently, lactulose is part of standard care in treatment of hepatic encephalopathy, as it can reduce the production of toxic ammonia due to its urease activity inhibiting function and its capability to improve nitrogenous product assimilation by gut microbes [103]. The use of prebiotics has been studied in IBD: notably, inulin has showed positive effects due to its capability of promoting the growth of lactobacilli and bifidobacteria and of inducing the production of short-chain fatty acids, with evidence of improved mucosal lesion scores and diminished mucosal inflammation in preclinical models [100]. Evidence of inulin in humans with IBD is limited, but inulin seems promising in reducing endoscopical and histological mucosal inflammation of the pouch after colectomy [104]. The use of prebiotics in IBS has been assessed in some randomized clinical trials, but, even if an amelioration of symptoms occurred, results were almost not significant or affected by biases [105]. Some selected dietary fibers seem to be able to stimulate a butyrate producing colonic ecosystem capable of preventing colorectal cancer by diminishing the rate of aberrant crypt foci formation in mice [106]. Further research will allow us to understand the potential role of prebiotics and synbiotics in other situations, as immunomodulation, obesity, cognitive defects, metabolic, and cardiovascular diseases [107].

Lastly, an emerging strategy that determines a hard reset of a disturbed microbial ecosystem is fecal microbiota transplantation (FMT), which consists of infusion of fecal material from a healthy donor into the gastrointestinal tract of a patient to heal a specific disease in a context of dysbiosis [108]. This strategy was used empirically in the 4th century in China to cure food poisoning and diarrhea [109] and was reintroduced in modern times in 1958, when Eiseman et al. [110] in Colorado performed fecal enemas to heal few patients with pseudomembranous colitis. Currently, the main worldwide accepted indication to FMT is multiply recurrent Clostridium difficile infection [111, 112], and it has been reported that this procedure can reduce disease complications, such as pseudomembranous colitis, bloodstream infections, length of hospitalization, and increase overall survival [113, 114].

In the setting of IBD, FMT showed some efficacy in inducing remission in patients with UC, but further studies are needed to define standardized procedures and outcome predictors [115]. Similarly, FMT may be beneficial for IBS if performed via colonoscopy instead of upper gastrointestinal tract, with apparently higher efficacy when transplanting donor stools rather than autologous stool [116]. The key factor of these procedures is introducing into the recipient a complete healthy gut microbiota and future perspectives involve metabolic disorders, cardiovascular disease, neuropsychiatric disorders, and malignancies treatment with promising results [117].

One of the first ideas of beneficial effects of bacterial administration derived from Elie Metchnikoff’s observation in 1905, who supposed that the longevity of the Bulgarian population was provoked by high lactobacilli dietary intake from dairy products [118]. Maybe, the initial use of probiotics in alimentary habits to improve health status was only part of cultural beliefs derived from empirical benefits, as it could have been the assumption of castor oil as a popular remedy for constipation [119]. Nowadays, thanks to the advent of new methods to study micro-organisms and to the possibility to perform in vivo and clinical studies, we know that an healthy gut microbiota is crucial for health and that perturbations of its equilibrium are associated with many intestinal and systemic diseases, even if many pathophysiological aspects remain still unknown. Restoring eubiosis, understanding how to modulate gut microbes in disease-specific settings, and elaborating standard protocols of microbiota-driven therapeutic strategies with definite outcomes predictors are the main challenges of researchers of the field. In recent decades, the discovery of gut microbiota could be somehow compared to a modern Pandora’s box, a container of secrets that was opened with curiosity and whose flow is hard to stop.

Declared none.

The authors have no conflicts of interest to declare.

This review received no external funding.

This work was contributed by all authors. The paper was discussed, designed, written, and reviewed by all authors.

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