Next-generation beneficial microbe: focus on the identification of Akkermansia muciniphila 

(source: Cani and de Vos, Frontiers Microbiology, Front Microbiol. 2017 Sep 22;8:1765.)

Akkermansia muciniphila is one of the most abundant single species in the human intestinal microbiota (0.5-5% of the total bacteria) and has been isolated and characterized as a mucin-utilizing specialist in 2004 by Muriel Derrien in her PhD research at Wageningen University (Derrien et al., 2004; Collado et al., 2007).  This discovery was initiated by the notion that the human body produces its own “prebiotics” or microbial substrates, namely mucus, an abundant glycoprotein that is specifically produced and degraded in the colon (Ouwehand et al., 2005). While germ-free mouse experiments showed that Akkermansia muciniphila showed immune and metabolic signaling, specifically in the colon, the exact functions of this unusual microbe remained enigmatic (Derrien et al., 2008; Derrien et al., 2011). Further indications for the function of Akkermansia muciniphila were subsequently determined in other prebiotic studies using inulin-type fructans that were initially characterized as bifidogenic compounds able to increase the abundance of Bifidobacterium spp. (Gibson and Roberfroid, 1995).

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Image : PD. CANI

Figure 1: Effects of A. muciniphila and derived products on host metabolism A. muciniphila has been found to be lower in several conditions such as during obesity, diabetes, intestinal inflammation, liver diseases or chronic alcohol consumption. This is associated with an altered gut barrier function leading to an increased plasma LPS levels and eventually triggering low grade inflammation and metabolic disorders. A. muciniphila alive or pasteurized as well as Amuc_1100 has been shown to restore gut barrier function likely by acting on TLR2 and restoring appropriate tight junction expression. All these results are associated with an increased mucus later thickness and an improvement of metabolic disorders. It is worth noting that an exploratory human investigation has shown that A. muciniphila is apparently safe.
AKkermansia• Science• Gut microbes• HOST and Beyond

Figure 1: Effects of A. muciniphila and derived products on host metabolism​ from: Cani and de Vos, Frontiers Microbiology, Front Microbiol. 2017 Sep 22;8:1765.

Thanks to the development of novel culture-independent techniques, we decided to revise in depth the impact of such kind of prebiotics on the overall microbial community in mice. Therefore, in search of potential novel bacterial candidates, we combined different techniques (phylogenetic microarray, high-throughput sequencing, gradient denaturation gel and qPCR), which allowed us to analyze and to compare all the bacteria that were present in the intestinal microbiota. The first surprise was to discover that more than 100 different taxa were affected by prebiotics (Figure 1) (Everard et al., 2011; Everard et al., 2014). Among these bacteria, we found that the relative abundance of Akkermansia muciniphila increased more than hundred-fold following the ingestion of prebiotics thereby reaching the abundance of up to 4.5% under high-fat diet (Everard et al., 2014), whereas this effect was lower under normal chow diet (0.09 to 2.5%) depending on the model (Everard et al., 2011; Everard et al., 2014). It is worth noting that these findings are confirmed in different set of experiments (Everard et al., 2013; Liu et al., 2016; Reid et al., 2016; Catry et al., 2017; Zhu et al., 2017). Interestingly, we and others discovered that Akkermansia muciniphila was less abundant in the intestinal microbiota of both genetic and diet-induced obese and diabetic mice (Everard et al., 2011; Everard et al., 2013; Everard et al., 2014; Schneeberger et al., 2015; Leal-Diaz et al., 2016; Ojo et al., 2016; Song et al., 2016; Singh et al., 2017), however, few studies reported in mice an increased abundance of Akkermansia muciniphila upon the ingestion of a high-fat high sucrose diet (Anhe et al., 2015; Carmody et al., 2015). It has also been largely demonstrated that inulin-type fructans feeding improves metabolic disorders associated with obesity, including a decreased fat mass, insulin resistance, lower liver steatosis and a reinforcement of the gut barrier (figure 1) (Cani et al., 2004; Cani et al., 2006; Cani et al., 2009; Maurer et al., 2010; Everard et al., 2011; Pachikian et al., 2012; Greer et al., 2016). Importantly, in humans the abundance of Akkermansia muciniphila was decreased in several pathological situations such as obesity, type 2 diabetes, inflammatory bowel diseases, hypertension and liver diseases (Png et al., 2010; Belzer and de Vos, 2012; Zhang et al., 2013; Dao et al., 2015; Yassour et al., 2016; Grander et al., 2017; Li et al., 2017). Conversely, antidiabetic treatments, such as metformin administration and bariatric surgery were both found to be associated with a marked increase in the abundance of Akkermansia muciniphila (figure 1) (Shin et al., 2014; Forslund et al., 2015; de la Cuesta-Zuluaga et al., 2017). Therefore, a large body of evidence suggested that Akkermansia muciniphila may contribute to protect from specific metabolic disorders and cardiometabolic risk factors associated with a low-grade inflammatory tone. 

Administration of Akkermansia muciniphila : multiple effects on the gut and beyond

Inspired by the numerous indications that the relative levels of Akkermansia muciniphila decreased during obesity and metabolic disorders in mouse and man, we decided to study the causal link between Akkermansia muciniphila and improvements in metabolism. This was done by investigating the impact of a daily oral supplementation with live Akkermansia muciniphila on the onset of obesity, diabetes and gut barrier dysfunction in mice. We found that the administration of live Akkermansia muciniphila at the dose of 2.10exp8 bacterial cells per day was partly protecting against diet-induced obesity in mice (Everard et al., 2013). Indeed, mice showed a 50% lower body weight gain when treated with live Akkermansia muciniphila without altering neither their dietary food intake nor the elimination of dietary fats in fecal matter. This protection was mirrored by two times less visceral and subcutaneous fat mass (Figure 1), but also by increased markers of fatty acid oxidation in the adipose tissue (Everard et al., 2013). In addition, animals receiving live Akkermansia muciniphila did no longer exhibited insulin resistance, nor infiltration of inflammatory cells (CD11c) in the adipose tissue, which is a key characteristic of obesity and associated low-grade inflammation (Everard et al., 2013). Interestingly, most of all the metabolic improvements observed following treatment with live Akkermansia muciniphila were in the range as those observed following oligofructose or inulin treatment (Cani et al., 2009; Dewulf et al., 2011; Everard et al., 2011; Everard et al., 2014), although live Akkermansia muciniphila was not affecting food intake behavior as do prebiotics like inulin and oligofructose. 

Knowing that these metabolic features can be caused by an increased plasma LPS level (i.e., metabolic endotoxemia) or bacterial translocation (Cani et al., 2007; Amar et al., 2011), we next investigated the gut barrier function by measuring several markers. We observed that live Akkermansia muciniphila prevented the development of metabolic endotoxemia, an effect associated with the restoration of a normal mucus layer thickness (Figure 1) (Everard et al., 2013). We also found that administration of live Akkermansia muciniphila restored the endogenous production of antimicrobial peptides. We then discovered that live Akkermansia muciniphila increased the endogenous production of specific bioactive lipids that belongs to the endocannabinoid family and are known to have anti-inflammatory activities and regulating the endogenous production of gut peptides involved in glucose regulation and gut barrier, respectively glucagon-like peptide-1 and 2 (GLP-1 and GLP-2) (Cani et al., 2016). It is worth noting that all these findings have subsequently been confirmed by different groups and extended to other specific disorders such as atherosclerosis, hepatic inflammation and hypercholesterolemia (Shin et al., 2014; Li et al., 2016; Shen et al., 2016; Grander et al., 2017; Plovier et al., 2017). 

Collectively all these data reinforce the assumption that live Akkermansia muciniphila can be considered as a next-generation beneficial microbe with the exceptional particularity that this bacterium can act on numerous facets of the metabolic syndrome and cardiometabolic disorders. Still, these discoveries have raised different fundamental questions that will still have to be studied in humans with the aim to generate new therapeutic tools.

Crossing the barrier of species: from mice to man

Akkermansia muciniphila requires specific culture conditions and complex animal-based medium (i.e., mucin from animal source) and although it may respire under microaerophilic conditions, the cells are relatively sensitive to oxygen (Ouwerkerk et al., 2016). These properties complicate the administration of Akkermansia muciniphila to human as to evaluate its potential, hence limiting its therapeutic perspectives. In order to solve this problem, a synthetic medium was developed in order to allow the culture of Akkermansia muciniphila with a high yield and devoid of compounds incompatible with administration in humans (Plovier et al., 2017)(Van der Ark et al., unpublished data). Besides the successful development of this synthetic medium, the previous assessment of the efficacy of Akkermansia muciniphila were performed with cells grown on a mucin-based medium. Therefore, the bacteria cultured on the different media were tested and compared. Interestingly, Akkermansia muciniphila retains its effectiveness independently of the medium used, and as previously observed, mice treated with the bacterium gained less weight, exhibited an improved glucose tolerance, and insulin resistance under hyperlipidic diet (Figure 1) (Plovier et al., 2017). 

Serendipity: the unexpected advantage of pasteurization

In 2013, it was showed that the protective effects of Akkermansia muciniphila disappeared when the bacterium was destroyed by using autoclaving, a heat treatment that destroyes all the constituents of bacteria and spores (Everard et al., 2013). As Akkermansia muciniphila is a Gram-negative bacterium and hence no spore-former, we were interested what the effects would be of pasteurization, a milder heat inactivation method than autoclaving. Therefore, we tested the impact of administrating pasteurized Akkermansia muciniphila (30 min at 70°C) cells on diet-induced metabolic disorders in mice. Unexpectedly, this method of inactivation did not abolish the effect of Akkermansia muciniphila but even exacerbated its beneficial impact. Specifically, mice receiving the pasteurized bacterium and the high-fat diet had a similar body weight gain and fat mass than those observed in mice fed a control diet. Again, these effects were independent of the food intake but pasteurized Akkermansia muciniphila increased the loss of energy in the feces of the treated mice, indicating a decrease in energy absorption that could contribute to explain the lower weight gain. Pasteurized Akkermansia muciniphila also strongly improved glucose tolerance, hepatic insulin sensitivity, and completely blocked the diet-induced metabolic endotoxemia. Although, the mechanisms of action of the bacteria are not yet fully elucidated, it is known that Akkermansia muciniphila express numerous highly abundant protein on its outer membrane (Ottman et al., 2017). Among these proteins, Amuc_1100, implicated in the formation of pili by Akkermansia muciniphila, was one of the most abundant (Plovier et al., 2017). 

Akkermansia muciniphila: a gate keeper that dialogues with the innate innate immune system 

We previously found that Akkermansia muciniphila was able to restore the expression of specific antimicrobial peptides (Everard et al., 2013). However, Nucleotide oligomerization domain (NOD)-like receptors (NLRs) and Toll-Like Receptors (TLRs) are a specialized group of membrane and intracellular proteins that play a critical role in the regulation of immunity and are directly involved in the recognition of bacterial constituents by the immune system. Therefore, we evaluated the potential of Akkermansia muciniphila to activate different NOD and TLRs. Strikingly, we found that the bacteria specifically interact with TLR2. TLR2 has been shown to modulate intestinal homeostasis and host metabolism (Caricilli et al., 2011; Brun et al., 2013), thereby participating in the interactions between microbes and host. In addition, to better characterize the interaction between Akkermansia muciniphila and this receptor, we took advantage of genomic and proteomic analyzes of the external membrane of the bacterium, which may be exposed to host receptors (Ottman et al., 2016). Among these proteins, Amuc_1100 was one of the most abundant. This protein is implicated in the formation of pili by Akkermansia muciniphila and thus could participate in the interaction between the bacterium and TLR2. This hypothesis was further confirmed by showing that a version of the genetically engineered protein (called Amuc_1100*) was effectively activating TLR2 and with the same magnitude as Akkermansia muciniphila. In addition, Amuc_1100* remained stable at the temperature used during pasteurization, and could therefore contribute to the effects of the pasteurized bacterium. Amuc_1100* was also able to replicate almost all the effects of Akkermansia muciniphila alive or pasteurized in high-fat diet fed mice. Akkermansia muciniphila, whether live or pasteurized, and Amuc_1100* also decreased high cholesterol levels induced by the high-fat diet. Conversely, the pasteurized bacterium specifically also reduced the triglyceridemia of the treated mice, reinforcing the idea that the pasteurization of Akkermansia muciniphila reinforces its protective effects. A potential mechanism explaining this could be the exposure of active molecules by the heat treatments, including Amuc_1100, or the inactivation of inhibitory compounds, or combinations thereof.

First assessment of Akkermansia muciniphila in humans with metabolic syndrome

As discussed earlier, Akkermansia muciniphila has various advantages as compared to other beneficial microbes and specific probiotics, at least in the context of the metabolic syndrome. Akkermansia muciniphila is present in the human milk, is highly abundant in lean and non-diabetic subjects, and is even highly increased upon metformin treatment of gastric bypass surgery, and this without obvious deleterious impact. This unique character does not preclude the fact the human investigations and safety assessment must be done. Hence, to become a putative future food supplement, the safety must be tested. We evaluated the toxicity and the emergence of possible side effects related to the administration of Akkermansia muciniphila in humans (20 subjects) as part of an ongoing clinical trial of individuals with metabolic syndrome (Plovier et al., 2017). To this end, we analyzed relevant clinical parameters related to liver, muscles and renal functions as well as markers of immunity and inflammation in individuals who received Akkermansia muciniphila daily for 2 weeks and then extended to 3 months. Whatever the formulation of Akkermansia muciniphila (live at 10exp9 and 10exp10 bacteria per day or pasteurized at 10exp10 bacteria per day), no changes were observed for the markers tested after 2 weeks or 3 months of daily administration. In addition, the frequency of side effects reported by patients were similar in the different groups. These first data indicate that Akkermansia muciniphila (active or pasteurized) is tolerated in individuals with metabolic syndrome and is likely not toxic.

While Akkermansia muciniphila is one of the handful of core microbes identified in the intestinal microbiota of over 1000 human adults (Shetty et al., 2017), the administration of its cells, either in live or pasteurized form, in a dietary supplement may be subject to regulatory frameworks that aim to safeguard the consumer. The regulatory requirements relating to the use of live Akkermansia muciniphila have recently been addressed (Gomez-Gallego et al., 2016). This review summarized the recent comprehensive studies related to Akkermansia muciniphila and its safety properties and provided criteria be addressed when Akkermansia muciniphila cells are to be considered as a novel food by the European Food Safety Authority in Europe.  One aspect that is relevant here and applies to other core intestinal microbes as well, is the fact that most if not all healthy subjects carry these anaerobes. So these have to be consumed at some stage and in this context it is important to note that A.mucinphila is present in early life microbiota and has been detected in mothers’ milk (Collado et al., 2007; Derrien et al., 2008; Collado et al., 2012; Jeurink et al., 2013; Ward et al., 2013). Another aspect relates to the antibiotic resistance of Akkermansia muciniphila that has been studied to some extent in healthy human subjects that carried high levels of Akkermansia muciniphila-like bacteria and apparently were sensitive to penicillin and tetracycline derivatives but resistant to vancomycin (Dubourg et al., 2013). This was confirmed in in vitro studies on the antibiotic resistance profile with the type strain AmucT (Ouwerkerk PhD Thesis Wageningen University 2016). Moreover, inspection of the genome sequence did not reveal antibiotic resistance genes that are linked to known genetically transferrable elements (Gomez-Gallego et al., 2016).

Conclusions 

Since its discovery in 2004, numerous studies have mostly linked the abundance of Akkermansia muciniphila with beneficial effects, and this although very few exceptions exist in specific non-physiological models (i.e., gnotobiotic models, specific immune double knock-out models) (Seregin et al., 2017). 

Nowadays, Akkermansia muciniphila is widely considered as a novel potential candidate to improve metabolic disorders associated with obesity, diabetes, liver diseases and cardiometabolic disorders. Indeed, its administration has been shown to profoundly reduce the development of such diseases. 

Other important steps towards the development of Akkermansia muciniphila as a next-generation beneficial microbe have been successfully reached. First, the discovery that Akkermansia muciniphila remained effective by being grown on a synthetic medium compatible with administration in humans. Second, the discovery that inactivation of the bacteria by pasteurization improved its effects, and thus its stability and potential shelf life. Third, the identification of a key mechanisms of interaction between Akkermansia muciniphila and its host via the identification of Amuc_1100, and last but not least; fourth, the demonstration that Akkermansia muciniphila may be safely administered in the human targeted population. 

Finally, the pasteurized bacteria and the identification and the isolation of bacterial constituents such as the relatively small 30-kDa Amuc_1100 open the door to putative development of drugs based on Akkermansia muciniphila-related product that could also target pathologies such as type 1 diabetes, inflammatory bowel diseases or diseases where the intestinal barrier function is compromised.