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Bacteroides, our most abundant gram-negative bacteria

 

17th April 2019 

Translated from the original article in Catalan.

 

What are Bacteroides ?

Bacteroides is the best-known genus of the most abundant gram-negative bacterial group within us, specifically in the intestine. They are up to 8·1010 per gram of stool. They are strict anaerobes, non-sporulated, non-mobile, with a form of rod with rounded tips (Figure 1). They are resistant to bile salts, at the concentration of 20% of the small intestine, and they have a good ability to use polysaccharides.

 

Fig1 Gerard F2.large

Figure 1. Electronic micrograph of cells of Bacteroides sp. D8 (Gerard et al 2007)

First of all, it should be noted that there are excellent revisions of Bacteroides, such as that of Wexler (2007), describing their beneficial aspects in the intestinal microbiota, which we will comment on here, as well as the toxic aspects and other characteristics.

Bacteroides live exclusively in the gastrointestinal tract of animals, and therefore they show great flexibility to adapt to the nutritional conditions of the intestinal environment. As commensals and mutualists, they establish long-term partnerships with the guests and provide them with benefits. The adaptation of these bacteria includes making modifications to this environment. For instance, many Bacteroides code for cytochrome bd oxidase, which can reduce oxygen concentrations, making it easier for them to grow as strict anaerobes, and at the same time, other bacteria of the usual microbiota also benefit from this (Wexler, Goodman 2017).

The most common substrates of these bacteria are the vegetable polysaccharides of the diet and of host’s mucus (Wexler, Goodman 2017). These carbohydrates are degraded and fermented, producing mainly short-chain fatty acids (SCFA). Bacteroides are the main producers of propionate in intestinal tract, and this acid is one of the beneficial SCFA, together with acetate and butyrate, because they are an energy source for colonocytes and contribute to maintenance of the correct glucose homeostasis and lipid metabolism (Ríos-Covián et al 2017). Bacteroides also remove side chains from bile salts, facilitating the return of bile acids to liver circulation. On the other hand, another beneficial aspect is that they exclude other possible pathogens as they colonize the intestinal tract and do not let others settle.

Due to the fact that the animal’s intestinal tract is the main habitat and environmental reservoir of Bacteroides, it is thought that there has been a symbiotic evolutionary relationship between these bacteria and the hosts (Troy, Kasper 2010). As in many other evolutionary cases, this mutual commensalism between microorganisms and hosts is almost a symbiosis, where virtually each of the organisms cannot live without the other.

As habitual residents of the intestine, the vast majority of Bacteroides are not harmful, on the contrary. Nevertheless, in conditions of metabolic imbalances such as diabetes or surgical patients, some of them are opportunistic and can be pathogens, and some have a certain resistance to antibiotics. In fact, B. fragilis, the most abundant species in the microbiota of healthy people, can give in these cases very serious infections and is the most important anaerobic pathogen bacterium in humans (Mancuso et al 2005). The abundance of B. fragilis is evident even because their bacteriophages are used as tracers of human faecal matter in water (Jofre et al 1995).

 

What kind of bacteria is Bacteroides ?

As detailed in the NCBI Taxonomy section, the genus Bacteroides is a bacterium of the Fibrobacter-Chlorobi-Bacteroidetes superphylum. We can see its phylogenetic relationship with other bacterial groups in Figure 2. Bacteroidetes phylum also includes Cytophaga, Flavobacter and Sphingobacter, in addition to the Bacteroidia class, which mainly includes the Bacteroidales order. This includes 2 families: the Bacteroidaceae and the Prevotellaceae. Besides Bacteroides, Prevotella is another of the best-known genera, which in fact was previously known as B. melaninogenicus.

 

Fig2 Bern 12862_2004_Article_146_Fig1_HTML

Figure 2. Phylogenetic tree of the bacterial groups (Bern, Goldberg 2005).

 

Bacteroides, some of the predominant in the human intestinal microbiota

The human intestinal microbiota, and from mammals in general, is very complex, but surprisingly, there are few phyla that predominate. Specifically, 98% of identified bacteria in humans (Figure 3) belong to 4 phyla: 64% Firmicutes, 23% Bacteroidetes, 8% Proteobacteria and 3% Actinobacteria. Therefore, Bacteroidetes are one of the most predominant bacteria in the intestinal microbiota. In fact, since Firmicutes are such a large and diverse phylum, which includes microbes as diverse as clostridial and lactic acid bacteria, it can be considered that Bacteroidetes, as a much more homogeneous group, are practically the predominant ones.

Fig3 brock 767 modif

Figure 3. Bacterial composition of the human colon deduced from the 16S rRNA obtained from 17242 sequences of faecal samples (Madigan et al 2012)

 

To see in depth the predominant species of the intestinal microbiota, very recently, a metagenomic and functional study of 737 genomes sequenced from bacterial isolates of faecal samples from 20 British and American adults (Forster et al 2019) has been done. 273 bacterial species have been detected, of which 105 had not been found before. As we can see (Figure 4), among the 20 dominant species there are 8 Bacteroides, plus 2 Parabacteroides, that is 10 Bacteroidales, signalled in green. Therefore, they are half of the majority species. The other 10 are 6 clostridial (Firmicutes, in blue), 3 are Actinobacteria (in yellow) and 1 is Proteobacteria (in orange).

Fig4 Forster 2019 Fig4

Figure 4. Major species of the human intestinal microbiota, detected with metagenomic data analyses (Forster et al 2019).

 

Although the microbiota is different in each person, at the strain level the individual microbiota is very stable. In a study with 37 healthy people (Faith et al 2013) about 200 strains of 100 different species have been found, and 60% of the strains remain for each person in a period of 5 years. Of those that remain, those of Bacteroidetes and Actinobacteria are the most stable.

In the same study (Faith et al 2013), gut microbiota of 6 people in the same family have been compared and it has been found that among the 75 most common bacterial species in the 6 persons, 18 are Bacteroidetes (24%): 11 Bacteroides, 3 Parabacteroides, AlistipesBarnesiella, Odoribacter and Butyricimonas. The only species of the 75 found in everybody is a Bacteroides: B. vulgatus.

The microbiota that accompanies us is changing throughout life (Figure 5). In fact, there are relatively few Bacteroides in the babies. However, these bacteria are already present among the few microbes of the placenta, where Proteobacteria predominate (Aagard et al 2014). After the birth, Bacteroides are increasing over the first months and years, mainly with the weaning and diet changes, as microbial diversity increases. Then, in adults Bacteroides are ones of the most abundant microbes (Gómez-Gallego, Salminen 2016).

Fig5 GomezGallego fig 1

Figure 5. Changes in the human microbiota throughout life (Gómez-Gallego, Salminen 2016).

 

Solid food intake in children, between 4 months and 1 year, causes a significant increase in Bacteroidetes (Figure 6). We see the great difference in the microbial composition from 118 day to 370. It is a pity that in this study (Koenig et al 2011) no more intermediate samples were took between these days, where little by little children go from porridge and a bit of cereals, to the ingestion of peas and other legumes, carrots, potatoes, etc. This increase in Bacteroidetes with solid food is surely related to the fact that Bacteroidetes are specialists in the breakdown of complex polysaccharides, and at the same time these compounds promote their growth. At the same time, there is a clear increase in the levels of AGCC, an enrichment of microbial genes associated with the use of carbohydrates, a greater biosynthesis of vitamins, and also an increase of xenobiotic degradation. Therefore, the role of Bacteroidetes seems primordial in the establishment and maintenance of the adult’s microbiota. Even though there are differences between individuals, once adult, microbial composition is quite stable throughout life, with certain variations depending on changes in diet or habitat or medication.

Fig6 Koenig fig 3

Figure 6. Metagenomic analysis of DNA sequences extracted from faecal samples of children (Koenig et al 2011).

 

Bacteroides in other mammals

The intestinal microbiota is present in all animals with a more or less developed digestive system. Apart from the insects, whose microbiota has been deeply studied (Engel, Moran 2013), the most studied in this aspect are mammals, of course. Their composition has been studied (Ley et al 2008), specifically in faecal samples of 106 individuals of 60 species of 13 different taxa, including human, other primates, herbivores, carnivores and omnivores.

Of the 17 bacterial phyla found, 65% were Firmicutes, 16% Bacteroidetes, 8% Proteobacteria and 5% Actinobacteria, among others. Therefore, the relevance of the Bacteroides is evident, and the proportions are similar to those mentioned above for humans. Regarding the majority group of Firmicutes, it is a pity that this work, like others, does not distinguish between different groups, especially among lactic acid bacteria and Clostridiales. Curiously in this work there is a greater presence of Bacteroides in primates and omnivores in general, and also in some herbivores, than in carnivores (Figure 7). In these there are very few Bacteroides, and instead there are more gamma-Proteobacteria, probably enterobacteria (Ley et al 2008).

Fig7 Ley fig S1A

Figure 7. Percentage of faecal samples sequences of different mammals assigned to the main different bacterial phyla (Ley et al 2008)

 

Different Bacteroidales are biomarkers of lifestyles

In the search for microbial taxa that could be biomarkers of diets or lifestyles, it has been seen that the biomarker more clearly related with people from rich western countries is the genus Bacteroides, whereas to the sub-Saharan ones it is Prevotella, another one of the same phylum. These two genera, together with some from the clostridia group, are the most abundant ones.

If the long-term majority diet is rich in animal proteins and fats, as in Western countries, Bacteroides predominates, and if the diet is rich in carbohydrates like in sub-Saharan countries, Prevotella prevails (Gorvitovskaia et al 2016).

 

What about Bacteroides in cases of dysfunction?

The beneficial relevance of Bacteroides, or their group, Bacteroidetes,on health is obvious in cases of diseases or dysfunctions such as allergies or obesity (Figure 8), where the diversity of the microbiota is much lower, and the number of Bacteroidetes is low.

Fig8 GomezGallego fig 2

Figure 8. Changes in the microbiota in dysfunctional situations such as allergies and obesity. (Gómez-Gallego, Salminen 2016).

 

Bacteroides against obesity

Well-known experiments of intestinal microbiota in relation to obesity have been those carried out with mice without previous microbiota colonized with microbiota from human twins of which one was obese and the other lean (Ridaura et al 2013). The result was that the mice with obese twin microbiota (Ob) became obese, while those of lean twin microbiota remained lean (Ln) (Figure 9). In addition, in the lean mice a greater intestinal production of SCFA and a greater microbial transformation of the bile acids were observed, whereas in the obese there was a greater metabolism of branched amino acids.

As mentioned in the previous section, in the obese mice a reduction of 50% Bacteroidetes is observed, apart from an increase in Firmicutes and methanogens (Figure 10). And as we see the Archaea methanogens decrease the hydrogen, producing methane, and the lower level of hydrogen promotes fermentation of ingested food in excess by the Firmicutes.

Fig9 mice obese lean Kay Chersnush

Figure 9. Obese and lean mice resulting from colonization with gut microbiota from obese and lean human twins respectively (image of Kay Chernush / Getty Images).

 

Fig10 brock 768 modif

Figure 10. Differences in intestinal microbial communities between lean (left) and obese (right) mice (Madigan et al 2012).

 

The most surprising, however, of this work (Ridaura et al 2013) is the cohabitation experiment of the two types Ob and Ln mice, where it is observed that after 10 days of coexisting together, the obese have diminished their body fat (Figure 11), and when their microbiota have been studied by sequencing, a transfer of the microbiota from lean mice to obese is observed (Figure 12). As we can see, the main bacteria transferred are Bacteroidales, which strengthens the importance of these bacteria.

Fig11 ridaura change body

Figure 11. Adiposity (% body fat) of obese (Ob) and lean mice (Ln), and the same after 10 days of cohabitation in the same cage (Obch and Lnch) (Ridaura et al 2013).

 

Fig12 ridaura ob ln bacteroi

Figure 12. Demonstration of the transfer of Bacteroidales (7 species: 5 Bacteroides, 1 Parabacteroides and 1 Alistipes) of the intestinal microbiota of lean mice (Lnch)  to the obese (Obch) after 10 days of cohabitation in the same cage. Each column corresponds to a mouse (Ridaura et al 2013).

 

Bacteroides against cholesterol

It has been known for many years that the intestinal microbiota is able to convert cholesterol in its saturated form, coprostanol (Figure 13). In other mammals some Eubacterium (belonging to the clostridial group) have been found to be responsible, but in humans we did not know what microorganisms could do it. Recently Gérard et al (2007) have isolated a strain of human stool that is able to do it and has been identified as Bacteroides, probably a species close to B. vulgatus.

Fig13 Gerard colesterol

Figure 13. Formulas of cholesterol and coprostanol (Gerard et al 2007)

 

Glycans (polysaccharides), important for mutualism between Bacteroides and the human host

Most non-digested macromolecules that reach the colon are glycans (word virtually synonymous of polysaccharides), which are a very important part of the fibre. The only glycan that is practically digested previously in the small intestine is starch. The consortium of microorganisms that inhabit the colon produces a huge enzymatic repertoire with the ability to degrade a range of complex polysaccharides that the host cannot process. That’s why the intestinal microbiota is often referred to as a metabolic organ.

On the other hand, the abundant commensal microbes of the intestinal microbiota must resist the inhospitable conditions of the previous sections and to settle in the colon without affecting the host. Therefore, instead of interacting with the epithelial cells of the intestine, they remain in the external mucus layer on the epithelial surface. At the same time, this mucus protects resident microbes from attacks by other bacteria and bacteriophages, and it is a nutrient substrate. It has been shown that the ability to survive in this ecosystem is closely related to the use and production of glycans by resident bacteria (Comstock 2009).

Well, precisely this ability to interact with glycans is an important characteristic of Bacteroidales, which, as we have seen, are the most abundant microorganisms in the intestine, along with Firmicutes. In fact, Bacteroidales have an extensive enzymatic machinery to use the complex polysaccharides present in the colon, and use them as a source of carbon and energy. This great capacity has been proven by sequencing the genome of B. thetaiotaomicron (Xu et al 2003) where it has found containing more than 80 loci of polysaccharides that encode proteins related to the detection, importation and degradation of specific glycans of the colon.

As we can see (Figure 14), Bacteroides use both the glycans of the host’s diet and those produced by the intestinal epithelium, they metabolize them, and produce the beneficial SCFA, and on the other hand, they synthesize glycans that accumulate in the form of exopolysaccharide (EPS) contributing to form biofilms, and in capsules that give immune signals to the host (Comstock 2009). All in all, the relevance of the glycans in the mutual relations between Bacteroides and the human host is confirmed.

Fig14 Comstock F1

Figure 14. Use and production of glycans (polysaccharides) by Bacteroides. IM (inner membrane): cytoplasmic membrane; OM (outer membrane): external part of the gram-negative cell wall; EPS: exopolysaccharide of mucosal layers, not covalently linked, unlike the capsular polysaccharide (Comstock 2009).

 

In addition to the glycans produced by the host, some Bacteroides can also use those that produce other microorganisms of the microbiota, as shown by B. fragilis, the most frequent species on the surface of the intestinal mucosa, which can metabolize exopolysaccharides produced by bifidobacteria (Ríos-Covian et al 2016). EPS production for bifidobacteria is stimulated by bile. This ability of B. fragilis to use EPS of bifidobacteria gives them more survival capacity when nutrients are scarce. At the same time, the degradation of the EPS can affect the viability of the bifidobacteria, and therefore, Bacteroidales would have a regulatory role of the intestinal microbiota in general.

Some glycans produced by Bacteroidales have a beneficial effect on the host’s immune system. In particular, it has been seen that polysaccharide A (PSA) produced by B. fragilis is able to activate the immune response on T-cells dependent, which influences the development and homeostasis of the immune system (Troy, Kasper 2010). In fact, the colonization of germ-free mice (without microbiota) with B .fragilis is sufficient to correct the previous imbalance of cells Th1 and Th2 (T helper) (Figure 15). In addition, PSA can protect against colitis, such as those produced by Helicobacter, by repressing proinflammatory cytokines associated with another type of T cells -Th17- and other mechanisms (Mazmanian et al 2008).

Fig15 Troy Fig1 PSA B fragilis

Figure 15. Impact of polysaccharide A (PSA) of Bacteroides fragilis in the development of the immune system by recovering the balance of Th1/Th2 cells (Troy, Kasper 2010).

 

The diet can make Bacteroides contribute to a good metabolic balance

In relation to said above about glycans such as EPS, it has been seen that if in the environment there is little organic nitrogen and an easily fermentable carbon source such as glucose, Bacteroides produce more lactate and less propionate, and instead with more organic nitrogen (yeast extract) and polysaccharides, these bacteria produce more propionate (Ríos-Covián et al 2017). When EPS are present, as more complex carbohydrates and slowly fermented, the carbon of the amino acids can be incorporated at the level of pyruvate, and then the path to succinate and propionate is enhanced and the redox equilibrium is maintained. Since a higher propionate production is beneficial to the host, these authors conclude that in cases of host metabolic dysfunctions, a good diet design (complex carbohydrates with organic nitrogen) would help to modify metabolic activity of Bacteroides, and these would help promote healthy effects to the host, in addition to interacting with the other beneficial bacteria.

 

Bacteroides as probiotics?

EFSA (European Food Safety Authority) has not accepted virtually any claim of positive effects of probiotics on health due to the restrictive requirements of studies with humans. The mechanism of probiotics action is strain-dependent and often is not well known. In addition, it could be that the incorporated bacteria did not produce sufficient measurable changes in healthy individuals to obtain a claim of health effect. Further studies at the genetic level, antibiotic resistance profile and probiotic selection criteria are required.

Traditional probiotics are mostly Lactobacillus and Bifidobacterium, but also some strains of other lactic acid bacteria, and from Bacillus, E. coli and Saccharomyces. Besides these, the so-called “next generation” probiotics are being introduced, thanks mainly to new culture and sequencing techniques. Among these new possible probiotics, there are the verrucomicrobial Akkermansia muciniphila, and some clostridia (see my post), like Faecalibacterium prausnitzii, the main producer of butyrate, but also some Bacteroidales. These ones also have a clear advantage over clostridia and other Firmicutes, because are much more stable in the intestinal tract throughout the life of the person (Faith et al 2013).

As we have seen, being some of the most abundant microorganisms in our intestinal microbiota, Bacteroides generally have clear benefits for the host, such as fighting against obesity, or cholesterol. Transplants of faecal microbiota for diarrhea associated with Clostridium difficile infections are being successful (Van Nood et al 2013) and therefore there is a clear possibility of using some specific strain or several ones, and in this way the Bacteroides are clear candidates due to their abundance in the samples of faecal microbiota.

In addition to those mentioned, other benefits of Bacteroides are those related to the immune system, at the level of cytokines and T cells and development of antibodies, in order to treat intestinal colitis, immune dysfunction, disorders of metabolism and even cancer prevention (Tan et al 2019).

Apart from the benefits shown to the host, a bacterial strain must have unambiguous security status in order to be considered probiotic. In the case of Bacteroides, recently, a strain (DSM 23964) of B. xylanisolvens isolated from stools of healthy humans has been studied and it has been shown to have no virulence determinants which have been found in some opportunistic Bacteroides, such as the enterotoxin bft and enzymatic biodegradative activities of extracellular matrix and PSA. This strain does not have resistance to antibiotics – although it is resistant to some – and no plasmids have been detected, which makes the transfer of possible resistance very unlikely. Therefore, this strain seems very safe (Ulsemer et al 2012a). It has also been seen that it does not adhere to the walls of the intestine, but it resists the action of gastric enzymes and low pH. In addition, as indicated by the name of the species, it degrades xylan and other pectins. These heteropolysaccharides are prebiotics, compounds that are beneficial for the gut microbiota.

Other basic probiotic characteristics found in this strain of B. xylanisolvens are the production of SCFA and immunomodulatory properties. These properties and the safety and good tolerance of this strain have been verified by incorporating it in fermented milk, after inactivation by heat. This milk has been ingested in trials by healthy humans, with safe effects (Ulsemer et al 2012b). Its safety has also been confirmed in studies of toxicity in mice, where high doses of the strain have not produced toxic or mutagenic effects, neither haematological nor histopathological damage (Ulsemer et al 2012c).

On the basis of these studies, the European Food Safety Authority has given the approval as a new food of the use of fermented milks with B. xylanisolvens DSM 23964 pasteurized (EFSA 2015). However, there is no claim to consider it as a probiotic, especially because bacteria are not viable as the product has been pasteurized, and by definition, probiotics should be living microorganisms.

 

Perspectives

We have seen the relevance of Bacteroides as one of the main components of the human intestinal microbiota and mammals in general. In addition to its fundamental role in the intestine and the possibilities of its use as a probiotic, it is an ideal model for the study of gut bacteria, because it is relatively easy of cultivating and has the potential to be genetically manipulated (Wexler, Goodman 2017). Therefore, it is necessary to deepen the knowledge of Bacteroidales, and in particular to know how they metabolize the host’s nutrients or mucus, or how they respond to changes in the host’s diet, or how they interact with the other microorganisms of the digestive tract. A better understanding of all these mechanisms will favour the design of therapeutics aimed at modifying the microbiota of patients suffering from various diseases and metabolic disorders linked to the intestinal microbiota (Wexler, Goodman 2017).

 

Bibliography

Aagaard K(2014) The placenta harbors a unique microbiome. Sci Transl Med 6, 237ra65

Bern M, Goldberg D (2005) Automatic selection of representative proteins for bacterial phylogeny. BMC Evolut Biol 5:34

Comstock LE (2009) Importance of glycans to the host – Bacteroides mutualism in the mammalian intestine. Cell Host & Microbe 5, 522-526

EFSA, European Food Safety Authority (2015) Scientific opinion on the safety of “heat-treated milk products fermented with Bacteroides xylanisolvens DSM 23964″ as a novel food. EFSA J 13(1):3956

Engel P, Moran NA (2013) The gut microbiota of insects – diversity in structure and function. FEMS Microbiol Rev 37, 699-735

Faith JJ et al (2013) The long-term stability of the human gut microbiota. Science 341, 1237439

Forster et al (2019) A human gut bacterial genome and culture collection for improved metagenomic analyses. Nature Biotechnol 37, 186-192

Gérard P et al (2007) Bacteroidessp. strain D8, the first cholesterol-reducing bacterium isolated from human feces. Appl Env Microbiol 73, 5742-5749

Gómez-Gallego C, Salminen S (2016) Novel probiotics and prebiotics: how can they help in human gut microbiota dysbiosis ? Appl Food Biotechnol 3, 72-81

Gorvitovskaia A et al (2016) Interpreting Prevotella and Bacteroides as biomarkers of diet and lifestyle. Microbiome 4:15, 1-12

Jofre J et al (1995) Potential usefulness of bacteriophages that infect Bacteroides fragilis as model organisms for monitoring virus removal in drinking water treatment plants. Appl Environ Microbiol 61, 3227-3231

Koenig JE et al (2011) Succession of microbial consortia in the developing infant gut microbiome. PNAS 108, 4578-4585

Ley RE et al (2008) Evolution of mammals and their gut microbes. Science 320, 1647-1651

Madigan MT, Martinko JM, Stahl DA, Clark DP (2012) Brock Biology of Microorganisms. 13th Ed. Pearson

Mancuso G et al (2005) Bacteroides fragilis – derived lipopolysaccharide produces cell activation and lethal toxicity via Toll-like receptor 4. Infect Immunity 73, 5620-5627

Mazmanian et al (2008) A microbial symbiosis factor prevents intestinal infammatory disease. Nature 453, 620-625

Ridaura VK et al (2013) Gut microbiota from twins discordant for obesity modulate metabolism in mice. Science 341, 1241214

Ríos-Covian et al (2016) Bacteroides fragilis metabolises exopolysaccharides produced by bifidobacteria. BMC Microbiol 16, 150

Ríos-Covian et al (2017) Shaping the metabolism of intestinal Bacteroides population through diet to improve human health. Front Microbiol 8, 376

Tan H et al (2019) Investigations of Bacteroides spp., towards next-generation probiotics. Food Res Internat 116, 637-644

Troy EB, Kasper DL (2010) Beneficial effects of Bacteroides fragilis polysaccharides on the immune system. Front Biosci 1, 15:25-34.

Ulsemer P et al (2012)a Preliminary safety evaluation of a new Bacteroides xylanisolvens isolate. Appl Env Microbiol 78, 528-535

Ulsemer P et al (2012)b Safety and tolerance of Bacteroides xylanisolvens DSM 23964 in healthy adults. Benef Microb 3, 99-111

Ulsemer P et al (2012)c Safety assesment of the commensal strain Bacteroides xylanisolvens DSM 23964. Regul Toxicol Pharmacol 62, 336-346

Van Nood E (2013) Duodenal infusion of donor feces for recurrent Clostridium difficile. New Eng J Medicine 368, 407-415

Wexler HA (2007) Bacteroides: the Good, the Bad, and the Nitty-Gritty. Clin Microbiol Rev 20, 593-621

Wexler AG, Goodman AL (2017) An insider’s perspective: Bacteroides as a window into the microbiome. Nat Microbiol 2, 17026

Wikipedia contributors. Bacteroides [Internet]. Wikipedia, The Free Encyclopedia, 2019 March 19

Xu J et al (2003) A genomic view of the human – Bacteroides thetaiotaomicron symbiosis. Science 299, 2074-2076

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A new probiotic modulates gut microbiota against hepatocellular carcinoma

24th August 2016

Over the last years the beneficial effects of the human intestinal microbiota on various health markers have been displayed, such as inflammation, immune response, metabolic function and weight. The importance of these symbiotic bacteria of ours has been proved. You can see these other posts related with our microbiota: “The good clostridia avoid us from allergies“, “Gut bacteria controlling what we eat” or “Good bacteria of breast milk

At the same time it has been seen that probiotics can be a good solution for many diseases with affected gut microbiota. Indeed, the beneficial role of probiotics to reduce gastrointestinal inflammation and prevent colorectal cancer has been proven.

However, recently it has been found that probiotics may have beneficial effects in other parts of the body beyond the gastrointestinal tract, particularly with immunomodulatory effects on an hepatocellular carcinoma (HCC). In this way, researchers at the University of Hong Kong, along with other from University of Eastern Finland, have published a study (Li et al, PNAS, 2016), where they have seen reductions of 40% in weight and size of HCC liver tumours in mice which were administered with a new mixture of probiotics, “Prohep.”

Hepatocellular carcinoma (HCC) is the most common type of liver cancer is the 2nd most deadly cancers, and it is quite abundant in areas with high rates of hepatitis. In addition, sorafenib, the drug most widely used to reduce the proliferation of tumour, is very expensive. The cost of this multikinase inhibitor is €3400 for 112 tablets of 200 mg, the recommended treatment of four pills a day for a month. Instead, any treatment with probiotics that would proved to be effective and could replace this drug would be much cheaper.

The new probiotics mix Prohep consists of several bacteria: Lactobacillus rhamnosus GG (LGG), Escherichia coli Nissle 1917 (ECN) and the whole inactivated by heat VSL#3 (1: 1: 1) containing Streptococcus thermophilus, Bifidobacterium breve, Bf. longum, Bf. infantis, Lb. acidophilus, Lb. plantarum, Lb. paracasei and Lb. delbrueckii.

In the mentioned work, Li et al. (2016) fed mice with Prohep for a week before inoculating them with a liver tumour, and observed a 40% reduction in tumour weight and size in comparison to control animals. As shown in Figure 1, the effect was significant at 35 days, and also for those who were given the Prohep the same day of tumour inoculation. Obviously, the effect of tumour reduction was much more evident when the antitumour compound Cisplatin was administered.

These researchers saw that tumour reduction was due to the inhibition of angiogenesis. This is the process that generates new blood vessels from existing ones, something essential for tumour growth. In relation to the tumour reduction, high levels of GLUT-1 + hypoxic were found. That meant that there was hypoxia caused by the lower blood flow to the tumour, since this was 54% lower in comparison to controls.

 

Fig 1 Li-Fig1B tumor size - days tumor

Figure 1. Change in tumour size. ProPre: administration of Prohep one week before tumour inoculation; ProTreat: administration of Prohep the same day of tumour inoculation; Cisplatin: administration of this antitumoral. (Fig 1B from Li et al, 2016).

 

These authors also determined that there was a smaller amount of pro-inflammatory angiogenic factor IL-17 and of Th17 cells of the immune system, cells also associated with cancer. The lower inflammation and angiogenesis could limit the tumour growth.

Moreover, these researchers established that the beneficial effects of probiotics administration were associated with the abundance of beneficial bacteria in the mice gut microbiota, analysed by metagenomics. So, probiotics modulate microbiota, favouring some gut bacteria, which produce anti-inflammatory metabolites such as cytokine IL-10 and which suppress the Th17 cell differentiation.

 

Fig 2 gut microbiota Eye of Science

Figure 2. Bacteria of the human intestinal microbiota seen by scanning electron microscope (SEM) (coloured image of Eye of Science / Science Source)

 

Some of the bacteria identified by metagenomics in the microbiota of mice that were administered with Prohep were Prevotella and Oscillibacter. The first is a bacteroidal, gram-negative bacterium, which is abundant in the microbiota of rural African child with diets rich in carbohydrates. Oscillibacter is a gram-positive clostridial, known in humans as a producer of the neurotransmitter GABA. Both are an example of the importance of some clostridial and bacteroidals in the gut microbiota. In fact, they are majority there, and although they are not used as probiotics, are found increasingly more positive functions, such as avoiding allergies (see “The good clostridia avoid us from allergies“).

It is known that these bacteria produce anti-inflammatory metabolites and therefore they would be the main involved in regulating the activity of immune cells that cause tumour growth. The observed reduction of tumour in these experiments with mice would be the result of combined effect of these administered probiotic bacteria together with the microbiota itself favoured by them. We see a potential outline of these actions in Figure 3.

Fig 3 Sung fig 2

Figure 3. Simplified diagram of the possible mechanisms of gut bacteria influencing on the polarization of Th17 cells in the lamina propria of the intestinal mucosa. The microbiota bacteria activate dendritic cells, which secrete cytokines (IL-22, IL-23, IL-27). The bacteria can promote Th17 immunity inducing IL-23, which can be involved by means of TLR ligands signal or extracellular ATP or serum amyloid A (SAA). Meanwhile, some probiotic strains could inhibit the development of Th17 by means of the production of IL-12 and IL-27, in addition to promoting the growth and colonization of the bacteria that induce Th17 (Sung et al 2012, Fig. 2).

 

Although we know that the cancer progression is a very complex process and that in the tumour microenvironments there is an infiltration of many different types of immune system cells, such as T cells, neutrophils, killer cells, macrophages etc, the Th17 helper cell subpopulation appears to be prevailing in the tumour progression, and therefore these effects of probiotics and microbiota open good prospects.

It is still early to say whether these findings will contribute to the treatment of human liver cancer, and therefore research in humans is needed, in order to see if these probiotics could be used as such or in tandem with some drug, depending on the tumour stage and size. In any case, all this opens a new range of possibilities for research of the molecular mechanisms of the beneficial effects of probiotics beyond the intestinal tract.

 

Bibliography

El-Nezami H (2016 april 27) HKU develops novel probiotic mixture “Prohep” that may offer potential therapeutic effects on liver cancer. The University of Hong Kong (HKU) 27 Apr 2016

El-Nezamy H, Lee PY, Huang J, Sung YJ (2015) Method and compositions for treating cancer using probiotics. Patent WO 2015021936 A1

Li J, Sung CYJ, Lee N, Ni Y, Pihlajamäki J, Panagiotou G, El-Nezami H (2016) Probiotics modulated gut microbiota suppresses hepatocellular carcinoma growth in mice. PNAS E1306-E1315

Oelschlaeger TA (2010) Mechanisms of probiotic actions – A review. Int J Med Microbiol 300, 57-62

Packham C (2016) Probiotics dramatically modulate liver cancer growth in mice. Medical Press, Med Research 23 Feb 2016

Silgailis M (2016) Treating some cancers with probiotics in the future ? Probiotic Prohep. Lacto Bacto: Health, Microbes and More 23 Feb 2016

Sung CYJ, Lee NP, El-Nezami H (2012) Regulation of T helper by bacteria: an approach for the treatment of hepatocellular carcinoma. Int J Hepatology ID439024, doi:10.1155/2012/439024

UEF News and Events (2016) A novel probiotic mixture may offer potential therapeutic effects on hepatocellular carcinoma. University of Eastern Finland 1 Mar 2016

 

We have good clostridia in the gut and some of them prevent allergies

21st March 2015

Clostridia: who are they ?

The clostridia or Clostridiales, with Clostridium and other related genera, are Gram-positive sporulating bacteria. They are obligate anaerobes, and belong to the taxonomic phylum Firmicutes. This phylum includes clostridia, the aerobic sporulating Bacillales (Bacillus, Listeria, Staphylococcus and others) and also the anaerobic aero-tolerant Lactobacillales (id est, lactic acid bacteria: Lactobacillus, Leuconostoc, Oenococcus, Pediococcus, Lactococcus, Streptococcus, etc.). All Firmicutes have regular shapes of rod or coccus, and they are the evolutionary branch of gram-positive bacteria with low G + C content in their DNA. The other branch of evolutionary bacteria are gram-positive Actinobacteria, of high G + C and irregular shapes, which include Streptomyces, Corynebacterium, Propionibacterium, and Bifidobacterium, among others.

 

flora_cover

 

Being anaerobes, the clostridia have a fermentative metabolism of both carbohydrates and amino acids, being primarily responsible for the anaerobic decomposition of proteins, known as putrefaction. They can live in many different habitats, but especially in soil and on decaying plant and animal material. As we will see below, they are also part of the human intestinal microbiota and of other vertebrates.

The best known clostridia are the bad ones (Figure 1): a) C. botulinum, which produces botulin, the botulism toxin, although nowadays has medical and cosmetic applications (Botox); b) C. perfringens, the agent of gangrene; c) C. tetani, which causes tetanus; and d) C. difficile, which is the cause of hospital diarrhea and some postantibiotics colitis.

 

clostridium_bacteria

Figure 1. The four more pathogen species of Clostridium. Image from http://www.tabletsmanual.com/wiki/read/botulism

 

Clostridia in gut microbiota

As I mentioned in a previous post (Bacteria in the gut …..) of this blog, we have a complex ecosystem in our gastrointestinal tract, and diverse depending on each person and age, with a total of 1014 microorganisms. Most of these are bacteria, besides some archaea methanogens (0.1%) and some eukaryotic (yeasts and filamentous fungi). When classical microbiological methods were carried out from samples of colon, isolates from some 400 microbial species were obtained, belonging especially to proteobacteria (including Enterobacteriaceae, such as E. coli), Firmicutes as Lactobacillus and some Clostridium, some Actinobacteria as Bifidobacterium, and also some Bacteroides. Among all these isolates, some have been recognized with positive effect on health and are used as probiotics, such as Lactobacillus and Bifidobacterium, which are considered GRAS (Generally Recognized As Safe).

But 10 years ago culture-independent molecular tools began to be used, by sequencing of ribosomal RNA genes, and they have revealed many more gut microorganisms, around 1000 species. As shown in Figure 2, taken from the good review of Rajilic-Stojanovic et al (2007), there are clearly two groups that have many more representatives than thought before: Bacteroides and Clostridiales.

 

Rajilic 2007 Fig 1

Figure 2. Phylogenetic tree based on 16S rRNA gene sequences of various phylotypes found in the human gastrointestinal tract. The proportion of cultured or uncultured phylotypes for each group is represented by the colour from white (cultured) passing through grey to black (uncultured). For each phylogenetic group the number of different phylotypes is indicated (Rajilic-Stojanovic et al 2007)

 

In more recent studies related to diet such as Walker et al (2011) — a work done with faecal samples from volunteers –, population numbers of the various groups were estimated by quantitative PCR of 16S rRNA gene. The largest groups, with 30% each, were Bacteroides and clostridia. Among Clostridiales were included: Faecalibacterium prausnitzii (11%), Eubacterium rectale (7%) and Ruminococcus (6%). As we see the clostridial group includes many different genera besides the known Clostridium.

In fact, if we consider the population of each species present in the human gastrointestinal tract, the most abundant seems to be a clostridial: F. prausnitzii (Duncan et al 2013).

 

Benefits of some clostridia

These last years it has been discovered that clostridial genera of Faecalibacterium, Eubacterium, Roseburia and Anaerostipes (Duncan et al 2013) are those which contribute most to the production of short chain fatty acids (SCFA) in the colon. Clostridia ferment dietary carbohydrate that escape digestion producing SCFA, mainly acetate, propionate and butyrate, which are found in the stool (50-100 mM) and are absorbed in the intestine. Acetate is metabolized primarily by the peripheral tissues, propionate is gluconeogenic, and butyrate is the main energy source for the colonic epithelium. The SCFA become in total 10% of the energy obtained by the human host. Some of these clostridia as Eubacterium and Anaerostipes also use as a substrate the lactate produced by other bacteria such as Bifidobacterium and lactic acid bacteria, producing finally also the SCFA (Tiihonen et al 2010).

 

Clostridia of microbiota protect us against food allergen sensitization

This is the last found positive aspect of clostridia microbiota, that Stefka et al (2014) have shown in a recent excellent work. In administering allergens (“Ara h”) of peanut (Arachis hypogaea) to mice that had been treated with antibiotics or to mice without microbiota (Germ-free, sterile environment bred), these authors observed that there was a systemic allergic hyper reactivity with induction of specific immunoglobulins, id est., a sensitization.

In mice treated with antibiotics they observed a significant reduction in the number of bacterial microbiota (analysing the 16S rRNA gene) in the ileum and faeces, and also biodiversity was altered, so that the predominant Bacteroides and clostridia in normal conditions almost disappeared and instead lactobacilli were increased.

To view the role of these predominant groups in the microbiota, Stefka et al. colonized with Bacteroides and clostridia the gut of mice previously absent of microbiota. These animals are known as gnotobiotic, meaning animals where it is known exactly which types of microorganisms contain.

In this way, Stefka et al. have shown that selective colonization of gnotobiotic mice with clostridia confers protection against peanut allergens, which does not happen with Bacteroides. For colonization with clostridia, the authors used a spore suspension extracted from faecal samples of healthy mice and confirmed that the gene sequences of the extract corresponded to clostridial species.

So in effect, the mice colonized with clostridia had lower levels of allergen in the blood serum (Figure 3), had a lower content of immunoglobulins, there was no caecum inflammation, and body temperature was maintained. The mice treated with antibiotics which had presented the hyper allergic reaction when administered with antigens, also had a lower reaction when they were colonized with clostridia.

 

fig 4 skefta

Figure 3. Levels of “Ara h” peanut allergen in serum after ingestion of peanuts in mice without microbiota (Germ-free), colonized with Bacteroides (B. uniformis) and colonized with clostridia. From Stefka et al (2014).

 

In addition, in this work, Stefka et al. have conducted a transcriptomic analysis with microarrays of the intestinal epithelium cells of mice and they have found that the genes producing the cytokine IL-22 are induced in animals colonized with clostridia, and that this cytokine reduces the allergen uptake by the epithelium and thus prevents its entry into the systemic circulation, contributing to the protection against hypersensitivity. All these mechanisms, reviewed by Cao et al (2014), can be seen in the diagram of Figure 4.

In conclusion, this study opens new perspectives to prevent food allergies by modulating the composition of the intestinal microbiota. So, adding these anti-inflammatory qualities to the production of butyrate and other SCFA, and the lactate consumption, we must start thinking about the use of clostridia for candidates as probiotics, in addition to the known Lactobacillus and Bifidobacterium.

 

fig 4 Cao b

Figure 4. Induction of clostridia on cytokine production by epithelial cells of the intestine, as well as the production of short chain fatty acids (SCFA) by clostridia (Cao et al 2014).

 

References

Cao S, Feehley TJ, Nagler CR (2014) The role of commensal bacteria in the regulation of sensitization to food allergens. FEBS Lett 588, 4258-4266

Duncan SH, Flint HJ (2013) Probiotics and prebiotics and health in ageing populations. Maturitas 75, 44-50

Rajilic-Stojanovic M, Smidt H, de Vos WM (2007) Diversity of the human gastrointestinal tract microbiota revisited. Environ Microbiol 9, 2125-2136

Rosen M (2014) Gut bacteria may prevent food allergies. Science News 186, 7, 4 oct 2014

Russell SL, et al. (2012) Early life antibiotic-driven changes in microbiota enhance 
susceptibility to allergic asthma. EMBO Rep 13(5):440–447

Stefka AT et al (2014) Commensal bacteria protect against food allergen sensitization. Proc Nat Acad Sci 111, 13145-13150

Tiihonen K, Ouwehand AC, Rautonen N (2010) Human intestinal microbiota and healthy aging. Ageing Research Reviews 9:107–16

Walker AW et al (2011) Dominant and diet-responsive groups of bacteria within the human colonic microbiota. The ISME J 5, 220-230

 

 

Bacteria in the gut are controlling what we eat

It seems to be so: the microbes in our gastrointestinal tract (GIT) influence our choice of food. No wonder: microbes, primarily bacteria, are present in significant amounts in GIT, more than 10 bacterial cells for each of our cells, a total of 1014 (The human body has about 1013 cells). This amounts to about 1-1.5 kg. And these bacteria have lived with us always, since all mammals have them. So, they have evolved with our ancestors and therefore they are well suited to our internal environment. Being our bodies their habitat, much the better if they can control what reaches the intestine. And how can they do? Then giving orders to the brain to eat such a thing or that other, appropriate for them, the microbes.

Imagen1Figure 1.Command centre of the gastrointestinal tract” (own assembly,  Albert Bordons)

Well, gone seriously, there is some previous work in this direction. It seems there is a relationship between preferences for a particular diet and microbial composition of GIT (Norris et al 2013). In fact, it is a two-way interaction, one of the many aspects of symbiotic mutualism between us and our microbiota (Dethlefsen et al 2007).

There is much evidence that diet influences the microbiota. One of the most striking examples is that African children fed almost exclusively in sorghum have more cellulolytic microbes than other children (De Filippo et al 2010).

The brain can also indirectly influence the gut microbiota by changes in intestinal motility, secretion and permeability, or directly releasing specific molecules to the gut digestive lumen from the sub epithelial cells (neurons or from the immune system) (Rhee et al 2009).

The GIT is a complex ecosystem where different species of bacteria and other microorganisms must compete and cooperate among themselves and with the host cells. The food ingested by the host (human or other mammal) is an important factor in the continuous selection of these microbes and the nature of food is often determined by the preferences of the host. Those bacteria that are able to manipulate these preferences will have advantages over those that are not (Norris et al 2013).

Recently Alcock et al (2014) have reviewed the evidences of all this. Microbes can manipulate the feeding behaviour of the host in their own benefit through various possible strategies. We’ll see some examples in relation to the scheme of Figure 2.

 

Fig 2 human microbiome behaviour appetite

Figure 2. As if microbes were puppeteers and we humans were the puppets, microbes can control what we eat by a number of marked mechanisms. Adapted from Alcock et al 2014.

 

People who have “desires” of chocolate have different microbial metabolites in urine from people indifferent to chocolate, despite having the same diet.

Dysphoria, id est, human discomfort until we eat food which improve microbial “welfare”, may be due to the expression of bacterial virulence genes and perception of pain by the host. This is because the production of toxins is often triggered by a low concentration of nutrients limiting growth. The detection of sugars and other nutrients regulates virulence and growth of various microbes. These directly injure the intestinal epithelium when nutrients are absent. According to this hypothesis, it has been shown that bacterial virulence proteins activate pain receptors. It has been shown that fasting in mice increases the perception of pain by a mechanism of vagal nerve.

Microbes can also alter food preferences of guests changing the expression of taste receptors on the host. In this sense, for instance germ-free mice prefer more sweet food and have a greater number of sweet receptors on the tongue and intestine that mice with a normal microbiota.

The feeding behaviour of the host can also be manipulated by microbes through the nervous system, through the vagus nerve, which connects the 100 million neurons of the enteric nervous system from the gut to the brain via the medulla. Enteric nerves have receptors that react to the presence of certain bacteria and bacterial metabolites such as short chain fatty acids. The vagus nerve regulates eating behaviour and body weight. It has been seen that the activity of the vagus nerve of rats stimulated with norepinephrine causes that they keep eating despite being satiated. This suggests that GIT microbes produce neurotransmitters that can contribute to overeating.

Neurotransmitters produced by microbes are analogue compounds to mammalian hormones related to mood and behaviour. More than 50% of dopamine and most of serotonin in the body have an intestinal origin. Many persistent and transient inhabitants of the gut, including E. coli, several Bacillus, Staphylococcus and Proteus secrete dopamine. In Table 1 we can see the various neurotransmitters produced by GIT microbes. At the same time, it is known that host enzymes such as amine oxidase can degrade neurotransmitters produced by microorganisms, which demonstrates the evolutionary interactions between microbes and hosts.

 

Table 1. Diversity of neurotransmitters isolated from several microbial species (Roschchina 2010)

Neurotransmitter Genera
GABA (gamma-amino-butyric acid) Lactobacillus, Bifidobacterium
Norepinephrine Escherichia, Bacillus, Saccharomyces
Serotonin Candida, Streptococcus, Escherichia, Enterococcus
Dopamine Bacillus, Serratia
Acetylcholine Lactobacillus

 

Some bacteria induce hosts to provide their favourite nutrients. For example, Bacteroides thetaiotaomicron inhabits the intestinal mucus, where it feeds on oligosaccharides secreted by goblet cells of the intestine, and this bacterium induces its host mammal to increase the secretion of these oligosaccharides. Instead, Faecalibacterium prausnitzii, a not degrading mucus, which is associated with B. thetaiotaomicron, inhibits the mucus production. Therefore, this is an ecosystem with multiple agents that interact with each other and with the host.

As microbiota is easily manipulated by prebiotics, probiotics, antibiotics, faecal transplants, and changes in diet, controlling and altering our microbiota provides a viable method to the otherwise insoluble problems of obesity and poor diet.

 

References

Alcock J, Maley CC, Aktipis CA (2014) Is eating behavior manipulated by the gastrointestinal microbiota? Evolutionary pressures and potential mechanisms. BioEssays 36, DOI: 10.1002/bies.201400071

De Filippo C, Cavalieri D, Di Paola M, Ramazzotti M, et al (2010) Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proc Natl Acad Sci USA 107:14691–6

Dethlefsen L, McFall-Ngai M, Relman DA (2007) An ecological and evolutionary perspective on human-microbe mutualism and disease. Nature 449:811-818

Lyte M (2011) Probiotics function mechanistically as delivery for neuroactive compounds: Microbial endocrinology in teh design and use of probiotics. BioEssays 33:574-581

Norris V, Molina F, Gewirtz AT (2013) Hypothesis: bacteria control host appetites. J Bacteriol 195:411–416

Rhee SH, Pothoulakis C, Mayer EA (2009) Principles and clinical implications of the brain–gut–enteric microbiota axis. Nature Reviews Gastroenterology and Hepatology 6:306-314

Roschchina VV (2010) Evolutionary considerations of neurotransmitters in microbial, plant, and animal cells. In Lyte M, Freestone PPE, eds; Microbial Endocrinology: Interkingdom Signaling in Infectious Disease and Health. New York: Springer. pp. 17–52

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