Monthly Archives: April 2019
Posted by Albert Bordons
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.
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.
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.
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).
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, Alistipes, Barnesiella, 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).
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.
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).
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.
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.
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).
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.
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).
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.
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.
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).
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.
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).
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