Clostridioides difficile (C. difficile, previously called Clostridium difficile) is a gram-positive, spore-forming, obligate anaerobe. Spores allow C. difficile to persist in environments, and to be spread from infected subjects. Under specific circumstances (e.g., antibiotic-driven dysbiosis), spores are driven to germination in the large bowel, and present in a vegetative form that leads to clinical infection (Clostridium difficile infection [CDI]). In the infection phase, C. difficile produces two toxins, enterotoxin A and cytotoxin B that both cause damage to colonocytes and trigger the inflammatory response, leading to a variety of clinical pictures, from mild colitis to pseudomembranous colitis and toxic megacolon. [1]

In recent years, CDI has become a considerable healthcare and economical burden in most countries. Studies from the United States report an incidence of nearly 453,000 cases and of nearly 29,000 CDIrelated deaths in 2011, while the incidence in Europe is 124,000 cases/year, with nearly 3,700 deaths/year. Increased morbidity, hospitalization length and mortality, contribute to the considerable economic burden of CDI, which accounted for nearly $ 5 billions in the US in 2011, and for nearly € 3.7 billions in Europe in 2013. [2, 3] These figures show that the CDI incidence has risen worldwide, for several reasons. First, the increased use of antibiotics, which are a known as risk factors for CDI development. Furthermore, the spreading of specific ribotypes (mainly the virulent ribotype 027, but also the 017 in Asia, the 018 in Italy, the 17,621 in Eastern European countries, 24,422 in Oceania) has let CDI clusters develop. Additionally, there was also an increased number of diagnoses, due to the development of highly sensitive diagnostic tests (e.g., PCR), and the risen awareness of CDI among healthcare professionals. Overall, the main cause of the overall increase in CDI incidence appears to be the increased rate of recurrences. From 2001 to 2012, the annual incidence of recurrent CDI has increased by nearly 189%, while the increase in overall CDI incidence in the same time period was nearly 43%. [2] As recurrent infection is less likely than first episode to be cured by antibiotics, it is associated with longer hospitalization, increased morbidity and mortality too.

Despite this increase in diagnoses, the misdiagnosis/underdiagnosis of CDI is still relevant, as observed in the EUCLID study.
This finding suggests that a considerable number of patients with CDI is still not diagnosed, increasing the risk of disease diffusion.

Nosocomial CDI, a community-acquired CDI, appear to differ for several characteristics. First, nosocomial patients are more likely to present with a severe clinical picture, while community patients can even be asymptomatic carriers, increasing the risk of CDI spreading. Moreover, community- based CDI is known to spread also among patients without standard risk factors.

Risk factors for C. difficile infection

Although the exact pathogenic pathways of CDI are not yet clarified, several risk factors have been identified over time. [4] Their knowledge is relevant as the management of modifiable risk factors is a prevention measure against CDI. Most relevant risk factors include older age, use of antibiotics, proton pump inhibitors, and others (Figure 1).

Therapeutic management of CDI 

Conventional treatment of CDI

Traditionally, metronidazole and vancomycin have been the most common treatment options for CDI, being used both as first line options, while only vancomycin was recommended, as tapered or pulsed regimen, to treat recurrent disease.[8]

However, in recent years CDI has become more cumbersome to treat. In particular metronidazole was shown to achieve lower cure rates than vancomycin, so that vancomycin has been preferred to metronidazole also in primary infection. Overall, also vancomycin is losing its efficacy, and the rates of recurrent disease have grown. Morover, hypervirulent strains of C. difficile have emerged, specifically the ribotype 027, which is less responsive to standard antibiotic therapy and is associated with more severe clinical pictures [8]

In recent years fidaxomicin, a narrow spectrum antibiotic, was shown to be superior than vancomycin in treating CDI recurrences. However, its high costs and the recent evidence of its inferiority compared with fecal microbiota transplantation (FMT) in treating recurrent CDI are potential limitations to its widespread use.[9]

Therapeutic microbiota modulators: probiotics and fecal microbiota transplantation 

Generally, probiotics are considered a reliable option to restore healthy gut microbiota after a dysbiotic event, e.g., antibiotic treatments. Overall, some probiotics are known to be effective against antibiotic-associated diarrhea (AAD), which is a common adverse event of antibiotic regimens.[10-12] In a metanalysis of 21 randomized trials, Saccharomyces boulardii decreased significantly the risk of AAD (risk ratio: 0.47).[11]

As CDI is basically a subgroup of AAD, the efficacy of probiotics in preventing CDI was then investigated. Recently, a Cochrane review has shown, in a meta-analysis of 23 trials, that probiotics are both safe and effective for preventing CDI.[13] However, only specific probiotics, including Saccharomyces boulardii, Lactobacillus casei, a mixture of L. acidophilus and Bifidobacterium bifidum, and a mixture of L. acidophilus, L. casei and L. rhamnosus, have been found to be effective in preventing primary CDI after antibiotic therapies. In particular, S. boulardii was effective in preventing CDI in a cohort of elderly hospitalized patients, with likely saving of money. Indeed, a Canadian study showed that the use of preventative probiotics was able to save $ 518/patient than usual care, and to reduce the risk of CDI.[11] However, further, larger studies are needed to confirm the role of specific probiotics in CDI prevention.

Based on this outstanding evidence, scientific societies have included FMT among the treatment options for recurrent CDI.[14, 15] FMT is also known to increase overall survival and decrease hospitalization length in patients with recurrent CDI.[16]
Although FMT has been increasingly standardized over years, is still underdiffused worldwide. Future microbiota-based approaches that will guarantee a widespread diffusion of FMT include capsulized FMT and microbiota-based drugs.

TUBERCULOSIS AND GUT MICROBIOTA

^{Eribo OA, du Plessis N, Ozturk M, et al. The gut microbiome in tuberculosis susceptibility and treatment response: guilty or not guilty? Cell Mol Life Sci 2020 ; 77 : 1497-509.}

5-10% of persons infected worldwide with Mycobacterium tuberculosis (MT) will progress to active TB. Recent research highlighted that gut dysbiosis induced by treatment could be involved in the disease development by compromising immune protection against MT. This review summarizes how the gut microbiota, lung immunity could be linked during the disease; and how the gut microbiota dysbiosis induced by the protracted anti-TB antibiotics treatment is involved to an increased susceptibility to MT re-infection or TB recrudescence after successful treatment cure. The authors also indicate that the gut microbiota biosignature might help recognizing healthy from active TB patients.

KETOGENIC DIET, GUT MICROBIOTA AND IMMUNE RESPONSES

^{Ang QY, Alexander M, Newman JC, et al. Ketogenic diets alter the gut microbiome resulting in decreased intestinal Th17 cells. Cell 2020 ; 181 : 1263-75.}

Very low-carbohydrate, high-fat ketogenic diet (KD) is used in refractory pediatric epilepsy, and some evidence supports KD use in diabetes and obesity but their metabolic and immune consequences remain unclear. The authors examined the impact of KD on human and mice gut microbiota via metagenomics and metabolomics and compared with high-fat diets impact: several bifidobacterial species were reduced, and an increase of Firmicutes/Bacteroidetes ratio induced by high-fat diet reversed. Increased plasma β-hydroxybutyrate levels inhibit bifidobacterial growth. KD reduced proinflammatory Th17 cell accumulation in mice adipose tissue and inhibited induction of intestinal Th17 cells.

THE ROLE OF SKIN MICROBIOTA IN ITCH

^{Kim HS, Yosipovitch G. The Skin Microbiota and Itch: Is There a Link? J Clin Med 2020 ; 9 : 1190.}

The authors discuss the role of skin microbiota in the pathogenesis of itch. Itch sensation is mediated via epidermal nerve fibres (pruriceptors) driving by chemical mediators that originate from a complex interaction between keratinocytes (KC), inflammatory cells, nerve endings and the skin microbiota, relaying itch signals to the brain. Skin dysbiosis is characterized by production of proteases, pathogen-associated molecular patterns, and toxins, leading to skin barrier damage. Mast cell degranulation induced by delta-toxin prompt inflammation and itching. Skin microbiota and brain communicate via neurochemicals (acetylcholine, histamine, catecholamines, corticotropin) originate from skin microbiota. Stress intensifies itch via the skin-brain axis, where the amygdala seems to modulate itching sensation via microbial signals. Chronic stress increases cortisol production, directly activates skin bacteria by increasing the virulence of skin pathogens, leading to a weakening of the skin barrier and to an aggravation of the itch sensation. The authors conclude that cosmetics/transdermal drugs that modulate skin microbiota might have the potential to ameliorate itch.

SKIN MICROBIOTA IN PSORIASIS

^{Quan C, Chen X-Y, Li X, et al. Psoriatic lesions are characterized by higher bacterial load and imbalance between Cutibacterium and Corynebacterium. J Am Acad Dermatol 2020 ; 82 : 955-61.}

The authors examined the microbiota in psoriatic lesions and unaffected skin in psoriasis vulgaris (PS) patients and healthy controls by quantitative PCR and 16S rRNA sequencing. Higher bacterial load and lower diversity was observed in PS lesions than patients unaffected and controls’ skin. Cutibacterium (Cu) was reduced in lesions, whereas Corynebacterium (Cr) was increased. Compared with patients’ unaffected skin, Cr/Cu + Cr ratio was higher in the lesions. These findings indicate that PS was the major cause for the imbalance between Cu and Cr between lesions and unaffected skin or controls. Cr load correlated with the severity of PS lesions, whereas Cu load showed correlation with the abnormity of skin capacitance. The present study suggests that skin microbiota might play a significant role in the pathogenesis of PS.

VAGINAL DYSBIOSIS AND RECURRENT IMPLANTATION FAILURE

^{Fu M, Zhang X, Liang Y, et al. Alterations in vaginal microbiota and associated metabolome in women with recurrent implantation failure. mBio 2020 ; 11 : e03242-19.}

Recurrent implantation failure (RIF) is defined as a failure to achieve a clinical pregnancy after transfer of at least four good-quality embryos in a woman under the age of 40 years. Embryonal and uterine factors or maternal systemic diseases may cause RIF, but some women do not have recognizable etiology. The authors focused on the vaginal microbiota and metabolome of women with RIF. They found that RIF patients suffered vaginal dysbiosis, having a more diverse and abundant bacteria with an increase of many anaerobic and aerobic bacteria which could be linked to bacterial vaginosis and aerobic vaginitis or urinary tract infection, respectively. Conversely, at genus level their vaginal microbiota was decreased in Lactobacillus (LB); at species level L. iners was reduced and L. crispatus was the most abundant species in the RIF group. Increased vaginal bacterial diversity, LB depletion and related metabolic changes could serve as biomarkers capable of predicting the risk of RIF.

THE ROLE OF VAGINAL MICROBIOTA IN URINARY TRACT INFECTIONS

^{Lewis A, Gilbert NM. Roles of the vagina and the vaginal microbiota in urinary tract infection: evidence from clinical correlations and experimental models. GMS Infect Dis 2020 ; 8 : Doc02.}

This review summarizes the role played by vaginal microbiota in urinary tract infections (UTI) as mounting evidence indicates that vagina may serve as a reservoir for uropathogens and increase susceptibility to UTI. Escherichia coli is the commonest cause of UTI and can colonize the vagina, which can be increased if the vaginal Lactobacillus (LB) colonization is reduced. Some vaginal bacteria are frequently detected in the urine but are underappreciated as uropathogens, because they are difficult to detect in routine clinical practice. For example, bacterial vaginosis (BV) is characterized by Gram-negative anaerobes, species belonging to Actinobacteria and Firmicutes phyla while LB is reduced and BV patients have a higher UTI risk. Gardnerella vaginalis is detected in BV and can cause acute or recurrent UTI. Group B Streptococcus may cause both aerobic vaginitis and UTI. Finally, some vaginal bacteria may enter the urinary tract and can transit briefly, cause immunomodulation or injury and unbalanced the host-pathogen interactions to influence the outcomes of uropathogenesis.

Microbiota and colorectal cancer

Third most frequent cancer in humans, sporadic colorectal cancer (CRC) develops following interactions between the host and its environment, and the IM is thought to be implicated [1]. Professor Sobhani presented the results of a study which investigated the links between epigenetic mechanisms promoted by bacteria of the IM and the onset of CRC [2]. Mice transplanted with faecal samples from patients with CRC developed precancerous colonic lesions, associated with an increase in hypermethylated genes. Donors with CRC exhibited methylation anomalies of several gene promoters associated with intestinal dysbiosis. Using the identified microbial and epigenetic signatures, a pilot study (n = 266) was conducted in humans in order to develop a blood test for the diagnosis of CRC. A cumulative methylation index (CMI, measuring the hypermethylation level of 3 genes) was identified as a predictive factor in the onset of CRC. These results were validated in a prospective cohort of 1,000 patients. Intestinal dysbiosis in patients with a positive CMI was characterised by an increase in pro-methylating bacterial species. This work indicates that intestinal dysbiosis associated with CRC could promote colon carcinogenesis via deregulation of the methylation of certain genes. The cumulative hypermethylation index (CMI) and/or pro-methylating bacteria are thus potential biomarkers for CRC diagnosis, or be used in the evaluation of the effects of treatments modulating the IM in patients with CRC.

A new dysbiosis marker in Crohn’s disease

In a study coordinated by Professor Seksik, the authors studied the role of MAM (microbial anti-inflammatory molecule, produced by Faecalibacterium prausnitzii and reduced in patients with Crohn’s disease, CD [3]) as a biomarker of intestinal dysbiosis and diagnostic aid in CD. The authors showed that loss of MAM is associated with the diagnosis of CD. This preliminary study in a small number of patients (24 patients in relapse, 24 in remission and 12 healthy controls) paves the way to the diagnosis of CD based on the IM, but these preliminary results require validation in independent cohorts.

A new therapeutic perspective in IBD

It is known that bacteria detect and respond to environmental signals (an ability called Quorum Sensing). Of the molecules which are part of this system, 3-oxo-C12:2 is low in patients with chronic inflammatory bowel disease (IBD), this reduction appears to be correlated with the observed intestinal dysbiosis [4]. In a study presented by D. Aguanno, the authors studied the impact of this molecule on the epithelial cells of the intestine and showed that this did not modify paracellular permeability but attenuated the deleterious effects on the tight junctions induced by pro-inflammatory cytokines. In a second study, Coquant et al. showed that 3-oxo-C12:2 exerted an anti-inflammatory effect on immunoreactive cells, partly mediated by the T2R138 receptor. This molecule may therefore have protective effects on the intestinal barrier, modulate the inflammatory response and thus represent a novel therapeutic perspective in IBD.

What do we already know about this subject?

The intestinal microbiota of neonates is characterised by lower bacterial diversity and a higher proportion of pathogenic bacteria. Moreover, their intestinal immune system is immature. These two factors modify the intestinal epithelial barrier and enhance the production of pro-inflammatory mediators.

Necrotising enterocolitis (NEC) is an ischemic and inflammatory disorder of the gastro- intestinal tract which affects premature neonates. The physiopathology of NEC remains poorly understood but intestinal dysbiosis is present, and also an inflammatory process. The use of antibiotics and antacids promote dysbiosis and also increases the risk of onset of NEC.

Neonates can also suffer from other ischaemic and inflammatory disorders, such as small bowel volvulus and localised gastro- intestinal perforations.

What are the main insights from this study?

This single centre pilot study profiled the composition of the FM and MM and also the mononuclear cells of the lamina propria and the pro-inflammatory cytokines of two groups of neonates: full-term or premature. Seven infants had extensive intestinal ischaemia (EII) (5 NEC, 1 small bowel volvulus and 1 total colonic ischaemia) and 7 infants had localised intestinal ischaemia (LII) (4 isolated perforations and 3 cases of intestinal atresia). The FM of 9 full-term infants was used as control. The MM of neonates with EII, compared with those with LII, contained: more Proteobacteria (p = 0.049) and fewer Bacteroidetes (p = 0.007) and Verrucomicrobia (p = 0.01) (Figure 1); fewer Bacteroides, Lachnospiracee, Ruminococcaceae and Akkermansia muciniphila (p < 0.05).

The FM was less diverse (Shannon index) in EII than in LII (p = 0.01). The relative abundance for the MM was similar between EII and LII for Proteobacteria and Firmicutes (p < 0.05). Similarly, a bacterial distribution with more Enterobacteriaceae in EII and more Ruminococcaceae, Bacteroides, Lachnospiracee and Staphylococcaceae in cases of LII.

The EII group exhibited increased numbers of lymphocytes B, T and NK (Natural Killer), in particular T CD3+ lymphocytes, TH17 (Figure 2A) and reduced numbers of T lymphocyte regulators (Tregs), with increased numbers of cells expressing TNFa (Figure 2B) and INFg (Figure 2C).

What are the consequences in practice?

This pilot study confirms that lack of bacterial diversity and the predominance of Enterobacteriaceae are risk factors for NEC, as is a reduction in numbers of Akkermansia muciniphila. Correction of this dysbiosis could modify the TH17/Tregs imbalance and reduce the production of mediators of inflammation (TNFa and INFg).

What do we already know about this subject?

Cholesterol is a key biological molecule that functions as a structural component of all animal cell membranes and is a pre-cursor of steroid hormones, vitamin D, and bile acids. Two main sources of cholesterol are thought to influence concentrations of this metabolite in serum: endogenous cholesterol synthesised in the liver and exogenous cholesterol derived from dietary components of animal origin (Figure 1). The cholesterol synthesised in hepatocytes is transported to the gallbladder and is then secreted into the small intestine along with other bile salts. In the intestine, biliary cholesterol (~1–2 g/day) mixes with dietary cholesterol (~0.2–0.4 g/day in the average American diet), and both sources are eventually transported into enterocytes for packaging into lipoprotein particles and secretion into the plasma. Hypercholesterolaemia is a risk factor for cardiovascular disease (CVD), which is the cause of one-fourth of all deaths in industrialised countries.

Reducing cholesterol transport in the intestine is a clinically validated strategy for lowering serum cholesterol levels. A range of gut microbes metabolise and modify dietary and host-derived molecules in the small intestine. Because both sources of cholesterol pass through this environment, the gut microbiota may influence serum cholesterol levels. Indeed, microbiota transfer from human donors with elevated serum cholesterol levels can impart this hypercholesterolaemia phenotype to mice. [2, 3] Other studies have reported that administering specific bacterial species can have cholesterol-lowering effects. [4] However, the precise mechanisms underlying these observations are currently unknown. The gut microbiota may exert cholesterol-lowering effects by metabolising intestinal cholesterol to coprostanol (Figure 1), which would reduce the amount of cholesterol absorbed from the intestine.

This microbiota-dependent transformation has been known to occur in humans since the early 1900s. Several coprostanol-generating gut bacteria with similar physical and biochemical characteristics have been reported from a variety of different sources including rats, baboons, and humans. However, most of these strains are not currently available and were never sequenced. Early work showed that coprostanol formation by this group of gut bacteria proceeds through an indirect reduction pathway involving the initial oxidation of cholesterol (1) to cholestenone (2), followed by reduction of the D4,5 double bond to form coprostanone (3), and subsequent re-reduction of ketone to generate coprostanol (4) (Figure 1). The bacterial enzymes responsible for this metabolism were never identified. More recently, other reports have implicated additional phylogenetically diverse gut bacteria in coprostanol formation. [5] While efforts to elucidate how gut microbial metabolism of cholesterol affects human serum cholesterol levels span over 100 years, mechanistic support for this connection has remained elusive due to a limited understanding of the gut microbes, genes, and enzymes responsible for coprostanol formation.

What are the main insights from this study?

The authors used a multi-disciplinary strategy to discover gut bacterial enzymes. This strategy, based on correlations between metagenomics and metabolomics data from existing human cohorts, identified and characterised an extensive family of cholesterol dehydrogenase enzymes from a clade of uncultured intestinal bacteria implicated in the metabolism of cholesterol to coprostanol. Firstly, the enzyme responsible for the first step in cholesterol transformation, called ismA, was identified in Eubacterium coprostanoligenes, a bacteria already known for this function. Analysis of sequencing data from human cohorts then identified homologous enzymes in a group of uncultured anaerobic bacteria.

The presence of these ismA genes in the microbiome was associated with the presence of coprostanol in stools and lower faecal cholesterol levels. Finally, to demonstrate the potential for these cholesterol- metabolising bacteria to influence human health, the authors showed that presence of ismA genes in human metagenomes is associated with a decrease in total cholesterol concentrations in serum that is on par with the effects observed from variants in human genes involved in lipid homoeostasis

What are the consequences in practice?

Overall, these findings confirm the role of gut-bacterial metabolism in modulating host cholesterol levels in the intestine and also, more importantly, on a systemic level. This work paves the way for the use of the gut microbiota as a predictive biomarker of high cholesterol and establishes the foundations for microbiota-targeted therapeutic interventions.

THE ONGOING THREAT OF RESPIRATORY VIRAL INFECTIONS

Viral respiratory tract infections are still a major concern worldwide as they lead to considerable socio-economic and health issues. Despite vaccine campaigns and antiviral medications, influenza - the commonly called “flu” - remains the most impacting in terms of infected individuals (5-10% of the global population) and deaths (> 600,000 per year). Along with annual epidemics, influenza is also responsible for pandemics every 10-20 years, the most famous being the 1918-1919 Spanish flu (50 million deaths) and the most recent, the 2009 H1N1 influenza pandemic (400,000 excess deaths) [1]. Pandemics are not restricted to influenza, as exemplified by the COVID-19 [2]. Of great concern is the increasing frequency of pandemics occurring over the last few decades, a phenomenon explained in part by climate changes and human practises, in general.

THE GUT MICROBIOTA IN HEALTH AND DISEASES

The gut microbiota has a crucial role in the maintenance of human health and is critical in the control of (respiratory) infections [3, 4]. Many factors can alter the diversity and composition of the gut microbiota, leading to dysbiosis. Among them, disease situations, such as infections and chronic inflammatory or metabolic disorders, can trigger profound alteration of the gut microbiota’s composition and function. Changes in intestinal bacterial communities can influence disease outcomes even in distant organs (including the lungs) [3, 4], as demonstrated by transfer experiments with dysbiotic microbiota. Below, we summarize the effects of acute viral respiratory infections on the gut microbiota.

VIRAL RESPIRATORY TRACT INFECTIONS LEAD TO GUT DYSBIOSIS

Clinical evidences suggest a gut microbiota dysbiosis during influenza infection. The relative abundance of Actinobacteria, Erysipelotrichea, Clostridia and beneficial butyrate producers (Lachnospiraceae and Ruminococcaceae families) is decreased in patients H1N1. On the other hand, opportunistic pathogens such as Escherichia- Shigella and Prevotella develop [5]. In experimental (mouse) models, transient gut dysbiosis also occurs with a peak at 5-7 days post-infection [6-9]. Changes occurred at taxonomic levels with no change in alpha diversity. Infection blunts the growth of health-promoting bacteria such as Lactobacilli, Bifidobacteria, and segmented filamentous bacteria. Many species capable of fermenting dietary fibres into short-chain fatty acids (SCFAs) are affected. In line, the production of SCFAs dramatically drops during influenza infection [9]. The reduced levels of beneficial commensals associate with the overgrowth of deleterious bacteria including Gammaproteobacteria (Escherichia coli) and mucus-degrading bacteria such as Verrucomicrobia (Akkermansia genus) and Ruminococcus. SARS-CoV-2 infection also triggers gut microbiota alterations in patients, including lower abundance of butyrate producers such as several genera from the Ruminococcaceae and Lachnospiraceae (Roseburia) families [5, 10]. On the other hand, a significantly higher relative abundance of opportunistic bacterial pathogens including Streptococcus (class Bacilli), Rothia and Actinomyces was observed. Of note, overgrowth of opportunistic fungal pathogens (Aspergillus and Candida spps) was also described in COVID-19 patients [11]. Collectively, viral respiratory infections lead to depletion of beneficial commensals and enrichment of opportunistic harmful pathogens. Putative changes in the gut microbiota’s structure, composition and functional activity might be biomarkers of disease severity.

MECHANISMS LEADING TO GUT DYSBIOSIS

There are probably several causes of gut dysbiosis during viral respiratory infections; these may include the release of inflammatory cytokines and the reduced food intake (Figure 1). Infection induces substantial weight loss due to a loss of appetite. Pair-feeding experiments in mice clearly indicate that a rapid fall in food intake mimics the changes in the gut microbiota observed during influenza infection [8]. Recent evidence suggests a role for TNFa in inappetence-associated dysbiosis during viral respiratory infection [12]. Type I and II interferons, which are essential for the host antiviral response, also play a part in gut dysbiosis [5,6].

Hypoxia (a feature of acute viral respiratory infection), alterations of the enteric nervous system and dysregulated local immune response are also likely to participate in gut dysbiosis [13] (Figure 1). In the case of COVID-19, along with these mechanisms, local viral replication is likely to play a role in gut dysbiosis. Angiotensin- converting enzyme II (ACE2), the receptor of SARS-CoV-2, is instrumental to maintain the gut’s microbial ecology. Considering the lack of available ACE2 during SARS-CoV-2 infection, one expects that this might influence the composition and functions of the gut microbiota [13].

CONSEQUENCES OF GUT DYSBIOSIS ON SECONDARY OUTCOMES

Gut dysbiosis during viral respiratory infection has local and distal consequences and might be an important contributor of disease severity and fatal outcomes (Figure 2). Patients experiencing viral respiratory infection can develop gastroenteritis- like symptoms such as abdominal pain, nausea, vomiting and diarrhoea. Alterations of the gut microbiota may explain these disorders. It is also likely that altered gut microbiota including the emergence of pathobionts and mucus degrading bacteria play a role in intestinal inflammation and disruption of the gut barrier integrity [6]. In turn, intestinal barrier leakage might increase endotoxin concentrations in the blood, ultimately triggering inflammation, cytokine overproduction and lung dysfunctions [14]. Acute viral respiratory infections can lead to secondary enteric infections and sepsis. Gut dysbiosis (and drop of SCFAs) might be important in this setting. Indeed, SCFAs play a key role in intestinal homeostasis, barrier integrity and control of enteric pathogens [15]. Along with local disorders, our recent data show that gut dysbiosis can remotely hamper host defense in the lungs [9] (Figure 2). In healthy conditions, the gut microbiota remotely arms the lungs against bacterial infection, in part by reinforcing the bactericidal activity of pulmonary macrophages [16].

During influenza, this axis is altered and opportunistic bacteria invade the lungs leading to bacterial superinfection, a major cause of death during influenza epidemics and pandemics [1]. We have shown that the reduced production of acetate (the major SCFAs) by the gut microbiota is partly responsible for this effect [9]. Collectively, dysbiosis might influence the gastrointestinal and pulmonary signs and symptoms (and overall mortality) of viral respiratory infections. Can we use the gut-lung pathway as a basis to better control the severity and mortality rate of viral respiratory infections?

INTERVENTIONAL PERSPECTIVES

The gut microbiota is vital in the lung’s defences against respiratory infection and interventional strategies that target intestinal commensals to preventively arm the lungs against viral pathogens and to protect the microbiota against the perturbations associated with viral infections are of major interest (Figure 3). This is particularly true in individuals with a general imbalance of the gut microbiota such as the elderly and individuals with co-morbidities, well known to be more susceptible to infections.

Approaches like (i) dietary ingredients intended to nourish our beneficial microorganisms (like prebiotic fibres) and (ii) indigenous bacteria (known as probiotics) to replenish our gut with missing beneficial microorganisms and to optimize their metabolic functions, might be relevant. These strategies, especially personalized (i.e. based on the analysis of the gut microbiota in the “at risk” population) might improve clinical outcomes and accelerate the recovery of patients that experience acute viral respiratory tract infections. Probiotics, including some Bifidobacteria and Lactobacillus spp, can decrease the severity of influenza infection, through still undetermined mechanisms [17]. Of highly topical concern, a recent study showed that oral bacteriotherapy in addition to the standard drug therapy showed promising clues in the management of COVID-19 patients [18].

The trillions of microorganisms that inhabit the human gut possess a greatly expanded set of genes compared to the host genome. Many of these genes encode protein-based catalysts, or enzymes, that enable gut microbes to perform a wide range of chemical reactions, expanding the chemistry associated with the human body. A hallmark of gut microbial metabolism is its variability; just as the composition of the microbiota differs between individuals, so too can the metabolic capabilities of this community. As we continue to identify associations between the gut microbiota and health and disease outcomes, it is becoming increasingly important to characterize microbial metabolic transformations at a molecular level.

One prominent activity associated with the gut microbiota is the ability to chemically modify the structures of small molecule drugs. [1] Orally administered drugs encounter gut microbes either prior to absorption in the small intestine or in the large intestine if they are poorly orally bioavailable. Orally administered or injected drugs, or drug metabolites, also reach the microbiota if they undergo biliary excretion into the intestine. Because a drug’s pharmacological activity directly arises from its chemical structure, microbial metabolism can have a large effect on drug action.

Effects of gut microbial drug metabolism

Gut microbial metabolism has various downstream consequences for drug action and efficacy (Figure 1). As the early examples of azo drugs illustrate, microbial metabolism of ‘prodrugs’ (inactive precursors) may be required to generate the active pharmacological agent. This knowledge has inspired the rational design of additional strategies for targeted drug release in the large intestine that rely on microbial metabolic activities Metabolism by the gut microbiota can also have negative effects on drug activity by disrupting interactions with intended host targets. One example is the natural product- based cardiac medication digoxin. In 5-10% of patients, the gut microbiota reduces the a, b-unsaturated lactone ring of digoxin to give dihydrodigoxin. This subtle modification, which is performed by the gut bacterium Eggerthella lenta, greatly reduces the binding affinity for digoxin’s target Na+/K+ ATPase, resulting in a loss of efficacy. [2] Another prominent example is the front-line Parkinson’s disease treatment L-dopa. Metabolism of L-dopa to dopamine by host enzymes in the brain is critical for alleviation of symptoms. Gut microbial metabolism of L-dopa also produces dopamine. [3,4] Because dopamine generated in the periphery cannot cross the blood brain barrier, this activity may reduce the amount of L-dopa that reaches the brain.

Finally, in addition to reducing activity, the chemical modifications installed by gut microbes can produce unwanted toxicity. For example, gut microbial metabolism was implicated in the lethality of co-administering the antiviral medication sorivudine with fluoropyrimidine chemotherapeutics. This outcome was traced to gut microbial metabolism of sorivudine to bromovinyluracil. This metabolite inhibits a key host enzyme involved in detoxifying 5-fluorouracil, increasing its concentration to lethal levels.

An important characteristic of gut microbial drug metabolism is its variability across patients. This phenomenon has its origins in the variability of the gut microbiota. Though some metabolic activities are found in many organisms, others are carried out by a small, low abundant subset of the gut community. Metabolism can vary between individual strains of the same species, as even closely related bacteria can have large differences in their genomes. It is therefore perhaps unsurprising that community composition is often a poor predictor of metabolism, and metabolism of individual drugs can be extensive in some individuals and absent in others. This variation likely has important but incompletely understood consequences for patients taking a range of small molecule drugs.

Understanding drug metabolism at a molecular level

In order to fully understand gut microbial drug metabolism, it is necessary to link individual activities with microbes, genes, and enzymes. Identifying specific drug-metabolizing microbes is typically needed to enable downstream mechanistic studies. This may be accomplished through screening available gut microbial isolates or isolating metabolizing organisms directly from complex gut microbiota samples. An important next step is connecting transformations of interest to genes and enzymes. This is crucial for studying metabolism in complex gut communities, as the genes encoding metabolic enzymes allow detection and prediction of individual activities in microbial genomes and microbiome sequencing data. Linking drug metabolism to microbial genes can be accomplished in multiple ways, including rationally searching genomes for enzymes with the requisite catalytic capabilities, using RNA-Seq to identify genes that are specifically upregulated in response to a drug, and using comparative genomics to associate genes with metabolic capabilities.

Identifying new metabolic activities

Until 2019, approximately 60 examples of gut microbial drug metabolism were reported. Two recent studies leveraged approaches from high-throughput screening and experimentation to perform large scale surveys of gut microbial drug metabolism, greatly expanding the scope of known transformations. Goodman and co-workers screened 76 human gut bacterial isolates for their ability to metabolize 271 small molecule drugs and found that two thirds of the drugs were depleted by at least one organism. [8] The Donia group performed an analogous screen of 575 drugs using a patient gut microbiome sample ex vivo and uncovered 45 new transformations. [6] These efforts suggest the scope of drugs subject to metabolism may be larger than previously known; however, the vast majority of these newly reported activities have not yet been confirmed in vivo, so their relevance for patients is unknown.

Manipulating gut microbial drug metabolism

Once the gut microbiota has been found to transform a small molecule drug, a logical next step is to ask how this activity may be controlled, both to assess the consequences of metabolism for drug action and to improve patient therapy should metabolism prove detrimental. Various methods have been employed to achieve this goal. Using gnotobiotic animal models (germfree animals colonized in a controlled manner with a defined microbiota), one can compare communities containing either drug metabolizing gut strains or deletion mutants missing specific activities. The utility of this approach was nicely illustrated by the Goodman lab’s studies of brivudine. [5]

However, genetic manipulation is challenging in native, complex microbial communities, prompting evaluation of alternative approaches. One potential strategy is to leverage knowledge of gut bacterial physiology to guide manipulation of the gut environment via dietary interventions. For example, digoxin Turnbaugh and co-workers noted that the presence of L-arginine downregulates drug metabolism by E. lenta. [2] They then showed that administering protein-rich diets to gnotobiotic mice colonized with E. lenta reduced drug inactivation in vivo.

Another exciting strategy is to identify small molecules that inhibit the activity of gut microbial drug metabolizing enzymes, as pioneered by the Redinbo lab in their studies of irinotecan metabolism. Irinotecan is a prodrug that is metabolized by host cells to the active topoisomerase inhibitor SN- 38. SN-38 is metabolized by the host via glucuronidation, which produces an inactive conjugate (SN-38G). This metabolite is excreted into the intestine, where the glucuronide is removed by gut bacterial b-glucoronidase (GUS) enzymes. This reactivation causes dose-limiting gastrointestinal tract toxicity. The Redinbo group used highthroughput screening to identify selective inhibitors of gut bacterial GUS enzymes, and found they prevented the severe side effects caused by irinotecan in a mouse model. [9] Subsequent work revealed that these compounds increase the efficacy of irinotecan by limiting its toxicity. [10] Together, this work has provided exciting proof-of-concept for therapeutically targeting gut bacterial metabolism and has prompted additional inhibitor discovery efforts.

Future frontiers

The successful development of GUS inhibitors as therapeutic candidates highlights one way in which gaining a molecular understanding of gut microbial drug metabolism could benefit patients. Another area that could be transformed by this knowledge is precision medicine. With an understanding of how specific therapeutics are metabolized by gut microbes, physicians could one day use microbiome sequencing data or microbiota-based diagnostic assays in deciding whether and how to prescribe particular medications.

Our growing appreciation of gut microbial drug metabolism may also influence the drug discovery process itself. Due to past associations with toxicity and side effects, many functional groups known to be transformed by gut bacteria are typically avoided by medicinal chemists. One could imagine uncovering new, unanticipated transformations early in drug development by screening individual gut microbes or complex patient communities for metabolism ex vivo, similarly to how drug candidates are typically tested for metabolism by host enzymes. Differences in gut microbiota composition and functions between animal models and humans should be taken into account in preclinical and clinical studies. Finally, it may be advisable to incorporate microbiome sample collection and analysis for drug metabolism into clinical trials. Correlating metabolism with differences in toxicity or efficacy might help in interpreting the results of such trials and defining target patient populations.

GUT MICROBIOTA AND THE INTESTINAL BARRIER

The gut microbiota is an initial barrier protecting the intestinal mucosa from pathogens. This complex ecosystem inhabits the gastrointestinal tract where it remains stable and limits access to the intestinal niches and to the nutrients required for the multiplication of exogenous bacteria by the phenomenon called “colonisation resistance” [1] (Figure 1). The enterocytes, which provide a physical barrier between the intestinal lumen and the host, absorb water and nutrients and secrete antimicrobial peptides, AMPs (RegIIIg, b-defensins and cathelicidin) [2]. By the recognition of microbe-associated molecular patterns, (MAMPs) by specific receptors (including the Toll-Like-Receptors, TLR), these cells will be able to transduce the signal to cytokines and chemokines thus signalling infection and recruiting immune cells (Figure 2). Paneth cells also participate in colonisation resistance by secreting AMPs (lysosyme, a-defensins, RegIIIg) [2]. The goblet cells – mucus-secreting – and the M cells have gatekeeping action, transporting antigens, intact and captured at random in the intestinal lumen arising from commensal bacteria or pathogens or dietary antigens. These will then be prepared by the dendritic cells (DC) and presented to the adaptive immune system. This function is vital to intestinal tolerance and the induction of mucosal immune responses [2]: there therefore is a constant balance between pro- and anti- inflammatory responses (Figure 2). In particular, this was demonstrated in mice models of induced colitis and in TLR receptor-deficient mice: the absence of microbiota or recognition of this reduces the proliferation of intestinal epithelial cells or barrier repair [2]. Lastly, the mucus also provides protection by capturing AMPs, which act to prevent the pathogens from reaching the epithelium. In the model of Muc2-deficient mice (Muc2 is the gene coding for one of the proteins making up the mucus), an increase in the translocation of commensal bacteria is observed and these animals develop intestinal inflammatory diseases [3].

CROSSTALK BETWEEN THE GUT MICROBIOTA AND THE INNATE IMMUNE SYSTEM

Among the players of the innate immune system which participate in intestinal homeostasis, antigen-presenting cells (APC), such as the macrophages (Mj) and the DCs have a major role. The Mj and the DCs synthesise IL-10 and thus promote differentiation of Treg [4] and the maturation of the Th17 lymphocytes via the implication of commensal bacteria: the segmented filamentous bacteria (SFB). These have the particular ability to adhere to the intestinal epithelial cells causing active stimulation of the immune system [5] (Figure 3). A study shows that colonisation of mice by these SFB, induces the differentiation of Th17 thus resulting in protection from Citrobacter rodentium (the murine equivalent of EPEC and EHEC). It has been suggested that this protection is due to the capacity of the SFB to cause Th17 to stimulate the synthesis of IL-22, a cytokine known to stimulate the synthesis of AMPs [6]. To come back to the DCs, these, by extending their dendrites between the epithelial cells, are able to phagocyte the bacteria present in the intestinal lumen. These commensal bacteria are then transported to the mesenteric lymph nodes to induce the production of IgA secreted by the plasma cells [1]. The innate lymphoid cells (ILC) also play an important role in intestinal homoeostasis; this is related to their capacity to initiate and direct intestinal immune responses. More specifically, the type 3 ILCs (ILC3) have a unique place in the interaction with the gut microbiota. By synthesising IL-22, these cells stimulate the production of mucus, AMPs and the secretion of chemokines and recruitment of polymorphonuclear (PMN) cells (Figure 2) [1].

CROSSTALK BETWEEN THE MICROBIOTA AND THE ADAPTIVE IMMUNE SYSTEM

The final maturation of the adaptive immune system is characterised by the colonisation of the intestinal mucosa by mature effector T-lymphocytes with inflammatory properties (Th17), T-lymphocytes with antiinflammatory properties (Treg) and B-lymphocytes (Figure 2). Besides effects on the macrophages and the differentiation of the Th17 cells, the SFB also stimulate the development of the lymphoid follicles and participate in the differentiation of the B-lymphocytes to IgA-producing plasma cells the action of which is the containment of pathogenic bacteria in the mucus [5]. Other commensal bacteria can stimulate adaptive immune responses: a mixture of 17 Clostridia strains isolated from a human faecal sample and introduced in mice induced an anti-inflammatory response by stimulating the Treg [7]. Faecalibacterium prausnitzii has also been identified for its anti-inflammatory action in vitro and in vivo by acting on the NF-kB factor, DCs and Mj which secrete IL-10 and enhance differentiation of Treg to the detriment of Th17 [8]. Of the Bacteroidetes, Bacteroides fragilis and B. thetaiotaomicron have also been described as exerting anti-inflammatory activity. B fragilis synthetises a polysaccharide A (PSA) that prevents pro-inflammatory IL-17 production and stimulates the anti-inflammatory of secretion IL-10 (Figure 3). In a specific model of Helicobacter hepaticus-induced colitis, PSA stimulated the development of lymphoid follicles, stimulated Treg lymphocyte cells and protected the mice [9].

MICROBIAL METABOLITES: IMPORTANT MEDIATORS IN THE CROSSTALK BETWEEN THE MICROBIOTA AND ADAPTIVE IMMUNITY

Short-chain fatty acids (SCFAs), tryptophan metabolites and bile salts are the principal metabolites produced by the gut microbiota which exert a protective effect against infections [9, 10]. Butyrate, propionate and succinate are known to act on intestinal homoeostasis, on mucus secretion, but also on the various cells of the immune system. Among other effects, butyrate has anti-inflammatory and anti- microbial effects. This action is exerted via the G-coupled protein receptors (GPR) found on the epithelial cells and the macrophages [9]. F prausnitzii produces large quantities of butyrate, which may partly explain its anti-inflammatory effect. It inactivates NF-kB and thus suppresses synthesis of the pro-inflammatory cytokines IFN-g, TNF-a, IL-1b, IL-8 by the enterocytes [8] (Figure 3) . It also induces metabolic and epigenetic modifications (via histone deacetylases, HDACs) macrophages in mice, thus amplifying their anti-microbial activities in vitro and in vivo [11]. Commensal bacteria can also metabolise tryptophan and produce antimicrobial substances. An example is the Lactobacilli, which utilise it as an energy source to synthesise an indole that binds to aryl hydrocarbon receptors (AhR) present on the ILC3. AhR triggers IL-22 secretion by the ILCs and this further drives the secretion of AMPs and protects against infections [9].

MICROBIOTA – INTESTINAL IMMUNE SYSTEM CROSSTALK FOR PROTECTION AGAINST VIRAL INFECTIONS

Among the enteric viruses, norovirus and rotavirus are the main causes of gastroenteritis [12]. The enteric viruses infect various cell types: enterovirus 71 specifically infect the goblet cells, whereas the rotavirus has a preferential tropism for the enterocytes [13] (Figure 4A). The gut microbiota acts as a barrier against enteric viral infections. The viruses have evolved and become adapted to their host, implementing mechanisms that enable them to cross the intestinal barrier and escape barrier immunity: it is in fact difficult to infect mice effectively with human enteric viruses by the oral route [13]. Virus penetration into the enterocyte triggers the secretion of type III interferon (IFN). Detection of a virus can induce IL-la, which activates the ILC3 to produce IL-22. This IL protects against enteric viral infections and acts synergistically with type III IFN to induce the expression of antiviral effectors and IL-15. Recognition of a virus by TLR-3 leads to the activation of the NF-kB pathway and to the production of IL-15 also. IL-15 activates the cytotoxic lymphocytes (NK cells). Those viruses, which have traversed the intestinal barrier, trigger the production of type I IFN by the macrophages of the lamina propria (Figure 4B). Some enteric viruses (rotavirus, reovirus, enterovirus) are able to adhere to the intestinal bacteria, enhancing penetration into the intestinal epithelial cells [13]. The SFB, which accelerate epithelial cell turnover produce protection against rotavirus infection in mice by expulsing infected cells [14]. The bile acids metabolised by the gut microbiota also act to protect the small intestine (but not the colon) from acute infection by norovirus in mice by enhancing the production of type III IFN in the small intestine [15].