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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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.
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.
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.
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.
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September 30th, 2015
The giant panda (Ailuropoda melanoleuca, literally Greek for “white and black cat feet”) is one of the most intriguing evolutionary mammal species. Despite its exclusively herbivorous diet, phylogenetically it is like a bear because it belongs to Ursids family, order Carnivores. Its diet is 99% bamboo and the other 1% is honey, eggs, fish, oranges, bananas, yams and leaves of shrubs.
It lives in a mountain area in central China, mainly in Sichuan province, and also in provinces of Shaanxi and Gansu. Due to the construction of farms, deforestation and other development, the panda has been driven out of the lowland where he lived. It is an endangered species that needs protection. There are about 300 individuals in captivity and 3000 in freedom. Although the numbers are increasing, it is still endangered, particularly due to its limited space (20,000 km2) and its very specific habitat (bamboo forests).
Thus, the giant panda has an almost exclusive diet of different species of bamboo, mainly the very fibrous leaves and stems, and buds in spring and summer. It is therefore a poor quality -digestive diet, with little protein and plenty of fibre and lignin content. They spend about 14 hours a day eating and can ingest about 12 kg of bamboo a day.
Most herbivores have modifications of the digestive tract that help them to retain the food in digestion process and contain microbial populations that allow them to eat exclusively plant materials, rich in complex polysaccharides such as cellulose and hemicellulose. These specializations may be compartmentalization of the stomach of ruminants and other typical non-ruminants (kangaroos, hamster, hippopotamus and some primates) or enlargement of the large intestine, characteristic of equines, some rodents and lagomorphs (rabbits and hares).
However, despite his exclusively herbivorous diet, surprisingly the giant panda has a typical carnivorous gastrointestinal tract, anatomically similar to dog, cat or raccoon, with a simple stomach, a degenerated caecum and a very short colon. The gastrointestinal tract of pandas is about 4 times the size of the body, such as other carnivores, whereas herbivores have about 10-20 times the size of the body, to efficiently digest large amounts of forage. With this, the panda intestinal transit time is very short, less than 12 hours. This severely limits the ability of potential fermentation of plant materials (Williams et al. 2013).
For these reasons, the digestion of bamboo for panda is very inefficient, despite their dependency. Pandas consume the equivalent of 6% of their body weight per day, with a 20% digestibility of dry matter of bamboo. Of this, 10% corresponds to the low protein content of bamboo, and the rest are polysaccharides, particularly with coefficients of digestion of 27% for hemicellulose and 8% for the pulp.
It seems as if the giant panda would have specialized in the use of a plant with high fibre content without having modified the digestive system, by means of an efficient chewing, swallowing large quantities, digesting the contents of cells instead of plant cell walls, and quickly excreting undigested waste (Dierenfield et al. 1982).
In addition, having a dependency on one type of plant such as bamboo can lead to nutritional deficiencies depending on seasonal cycles of the plant. In this regard, recently Nie et al. (2015) have studied the concentrations of calcium, phosphorus and nitrogen from different parts of the bamboo that a population of free pandas eat. They have seen that pandas in their habitat have a seasonal migration in two areas of different altitudes throughout the year and that fed two different species of bamboo. Both species have more calcium in the leaves and more phosphorus and nitrogen in the stems. As the seasonal variation in appearance and fall of leaves of two species is different due to the different altitude, when pandas are in one of the areas eat the leaves of a species and stems of the other while they do the reverse when they are in the other zone. So, pandas synchronize their seasonal migrations in order to get nutritionally the most out of both species of bamboo.
Another drawback of the bamboo dependence is flowering. It is a natural phenomenon that happens every 40-100 years, and when bamboo flowers, it dies, reducing the availability of food for pandas. During 1970-1980 there were two large-scale blooms in the habitat of pandas, and there were more than 200 deaths for this reason. However, and given that probably pandas have found during their evolution with many other massive blooms, in these occasions they are looking for other species of bamboo or travel long distances to meet their food needs (Wei et al. 2015).
In return, and as adaptation to eat this so specific food, the giant panda has a number of unique morphological features, such as strong jaws and very powerful molars, and especially a pseudo-thumb, like a 6th finger, which is actually a modified enlarged sesamoid bone, as an opposable thumb, which serves to hold bamboo while eating (Figure 1).
Figure 1. The “pseudo-thumb” of giant panda. Image from Herron & Freeman (2014).
And how is that the panda became an herbivore ?
It has been estimated that the precursor of the giant panda, omnivorous as other Ursids, began to eat bamboo at least 7 million years ago (My), and became completely dependent on bamboo between 2 and 2.4 My. This dietary change was probably linked to mutations in the genome, leading to defects in the metabolism of dopamine in relation to the appetite for meat, and especially the pseudogenization of Tas1r1 gene (Figure 2) of umami taste receptor (Jin et al. 2011). The umami is one of the five basic tastes, along with sweet, salty, sour and bitter. Umami is like “pleasant savoury taste”, usually recalls meat, and is related to L-glutamic acid, abundant in meat. This mutation in pandas favoured the loss of appetite for meat and reinforced their herbivore lifestyle. However, other additional factors had probably been involved, since Tas1r1 gene is intact in herbivores such as horses and cows (Zhao et al. 2010).
Figure 2. Phylogenetic tree of some carnivores with data for giant panda deduced from fossils (in blue) and from the molecular study of TasTr1 gene made by Zhao et al. (2010).
The intestinal microbiota of giant panda
As expected, when sequencing the complete genome of the giant panda (Li et al. 2010), specific genes responsible for the digestion of cellulose and hemicellulose have not been found. Logically, these complex polysaccharides of bamboo fibres would be possibly digested by cellulolytic microorganisms of the intestinal tract. So, their presence in panda must be studied.
When studying the sequences of 16S ribosomal DNA from faecal microbiota of various mammals, an increase in bacterial diversity is generally observed in sense carnivores – omnivores – herbivores (Ley et al. 2008). This diversity is lower in the panda than in herbivores, and as shown in Figure 3, pandas are grouped with carnivores (red circles) despite being herbivorous from the diet point of view.
Figure 3. Principal component analysis (PC) of faecal bacterial communities from mammals with different colours according to the predominant diet (Law et al. 2008)
The intestinal microbiota of most herbivores contains anaerobic bacteria mainly from groups of Bacteroides, Clostridials, Spirochetes and Fibrobacterials, that have enzymatic ability to degrade fibrous plant material and thus provide nutrients for its guests. Instead, omnivores and carnivores have a particularly dominant microbiota of facultative anaerobes, such as Enterobacteriaceae, besides some Firmicutes, including lactobacilli and some Clostridials and Bacteroides.
As for the giant panda, the first studies made with culture-dependent methods and analysis of amplified 16S rRNA genes (Wii et al. 2007) identified Enterobacteriaceae and Streptococcus as predominant in the intestinal microbiota. Therefore, this study suggests that the microbiota of panda is very similar to that of carnivores, as we see in the mentioned comparative study with various mammals (Law et al. 2008), and therefore with little ability to use cellulose or hemicellulose.
However, a later study done with sequencing techniques of 16S (Zhu et al. 2011) from faecal samples of 15 giant pandas arrived at very different conclusions and it seemed that they found the first evidence of cellulose digestion by microbiota of giant panda. In 5500 sequences analysed, they found 85 different taxa, of which 83% were Firmicutes (Figure 4), and among these there were 13 taxa of Clostridium (7 of them exclusive of pandas) and some of these with ability to digest cellulose. In addition, in metagenomic analysis of some of the pandas some putative genes for enzymes to digest cellulose, xylans and beta-glucosidase-1,4-beta-xilosidase for these Clostridium were found. Altogether, they concluded that the microbiota of the giant panda had a moderate degradation capacity of cellulose materials.
Figure 4. Percentage of sequences of the main bacterial groups found in faecal samples from wild individuals of giant panda (W1-W7) and captive (C1-C8), according to Zhu et al. (2011). Under each individual the n. sequences analysed is indicated.
But just three months ago a work (Xue et al. 2015) has been published that seems to go back, concluding that the intestinal microbiota of the giant panda is very similar to that of carnivores and have little of herbivores. It is an exhaustive study of last-generation massive sequencing of 16S rRNA genes of faecal samples from 121 pandas of different ages over three seasons. They obtained some 93000 sequences corresponding to 781 different taxa.
They found a predominance of Enterobacteriaceae and Streptococcus (dark red and dark blue respectively, Figure 5A) and very few representatives of probable cellulolitics as Clostridials. Moreover, these are not increased when more leaves and stems of bamboo are available (stage T3). These results correspond with what was already known of the low number of genes of cellulases and hemicellulases (2%), even lower than in the human microbiome. This negligible contribution of microbial digestion of cellulose, together with the commented fact that the panda is quite inefficient digesting bamboo, contradicts the hypothetical importance of digestion by the microbiota that had suggested a few years earlier, as we have seen before.
In addition, in this work a lot of variety in composition of microbiota between individuals has been found (Figure 5 B).
Figure 5. Composition of the intestinal microbiota from 121 giant pandas, with (A) the dominant genera in all samples and (B) the relative contribution of each individual dominant genera, grouped by age and sampling time (Xue et al. 2015).
In this paper, a comparative analysis between the compositions of the intestinal microbiota of giant panda with other mammals has been made, and it has confirmed that the panda is grouped again with carnivores and is away from herbivores (Figure 6).
Figure 6. Principal component analysis (PCoA) of microbiota communities from faecal samples of 121 giant pandas (blank forms), compared with other herbivores (green), omnivores (blue) and carnivores (red). The different forms correspond to different works: the circles are from Xue et al. (2015), where this Figure has been obtained.
All in all, the peculiar characteristics of the giant panda microbiota contribute to the extinction danger of this animal. Unlike most other mammals that have evolved their microbiota and digestive anatomies optimizing them for their specific diets, the aberrant coevolution of panda, its microbiota and its particular diet is quite enigmatic. To clarify it and know how to preserve this threatened animal, studies must be continued, combining metagenomics, metatranscriptomics, metaproteomics and meta-metabolomics, in order to know well the structure and metabolism of gut microbiota and its relationship with digestive functions and the nutritional status of the giant panda (Xue et al. 2015).
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2nd May 2015
The vine growers believe that the land on which they grow vines gives the wines a unique quality, and that is called terroir. We can consider that the physiological response of the vines to the type of soil and climatic conditions, together with the characteristics of the variety and form of cultivation, result in a wine organoleptic properties that define their terroir (Zarraonaindia et al 2015 ). However, it is not known if there could be a very specific microbiota of each terroir, as this subject has been barely studied.
Wine microorganisms in the grapes? Saccharomyces is not there or it has not been found there
The main protagonists of wine fermentations, alcoholic one (yeast Saccharomyces cerevisiae) and malolactic one (lactic acid bacteria Oenococcus oeni) usually do not appear until the must grape is fermenting to wine, in the cellar. In normal healthy grapes, S. cerevisiae is hardly found.
Oenococcus oeni in the grapes ? We have found it !
Regarding O. oeni, so far very little has been published about its presence and isolation from the grapes. In some works, as Sieiro et al (1990), or more recently Bae et al (2006), some lactic acid bacteria (LAB) have been isolated from the surface of grapes, but not O. oeni. Only Garijo et al (2011) were able to isolate a colony (only one) of O. oeni from Rioja grapes. Moreover, DNA of O. oeni has been detected in a sample of grapes from Bordeaux (Renouf et al 2005, Renouf et al 2007) by PCR-DGGE of rpoB gene, although in these works no Oenococcus has been isolated.
I am pleased to mention that recently our team have managed to isolate O. oeni from grapes, and typify them, and we are now working on a publication about it (Franquès et al 2015). Indeed, our research team of lactic acid bacteria (BL-URV), together with colleagues working on yeasts from the same group “Oenological Biotechnology” (Faculty of Oenology at the Universitat Rovira i Virgili in Tarragona, Catalonia, Spain) is working on a European project, called “Wildwine “(FP7-SME-2012 -315065), which aims to analyse the autochthonous microorganisms of Priorat area (South Catalonia), and select strains with oenological potential. This project also involves the Priorat Appellation Council and the cellar Ferrer-Bobet, as well as research groups and associations wineries from Bordeaux, Piedmont and Greece. In the framework of this project we took samples of grapes (Grenache and Carignan) from several vineyards of Priorat (Figure 1), as well as samples of wines doing malolactic fermentation. From all them we got 1900 isolates of LAB. We optimized isolation from grapes from the pulp and juice with various methods of enrichment, and so we got 110 isolated bacteria from grapes, identified as O. oeni by specific molecular techniques. Once typified, we have found that the molecular profiles of these strains do not coincide with commercial strains and so they are autochthonous. In addition, some of these strains from grapes were also found in the corresponding wine cellars.
Figure 1. Taking samples of Grenache (left) and Carignan (right) in Priorat area to isolate lactic acid bacteria such as Oenococcus (Pictures Albert Bordons).
The microbiota of grapes
The grapes have a complex microbial ecology, including yeasts, mycelial fungi and bacteria. Some are found only in grapes, such as parasitic fungi and environmental bacteria, and others have the ability to survive and grow in wines: especially yeasts, lactic acid bacteria (LAB) and acetic acid bacteria. The proportion of all them depends on the maturation of the grapes and the availability of nutrients.
When the fruits are intact, the predominant microbiota are basidiomycetous yeasts as Cryptococcus and Rhodotorula, but when they are more mature, they begin to have micro fissures that facilitate the availability of nutrients and explain the predominance just before the harvest of slightly fermentative ascomycetes as Candida, Hanseniaspora, Metschnikowia and Pichia. When the skin is already damaged more damaging yeasts may appear, as Zygosaccharomyces and Torulaspora, and acetic acid bacteria. Among the filamentous fungi occasionally there may have some very harmful as Botrytis (bunch rot) or Aspergillus producing ochratoxin. Although they are active only in the vineyard, their products can affect wine quality.
On the other hand, environmentally ubiquitous bacteria have been isolated from the grapes skin, as various Enterobacteriaceae, Bacillus and Staphylococcus, but none of them can grow in wine (Barata et al 2012).
Coming back to the possible specific microbiota of terroir, it has been found that some volatile compounds contributing to the aroma of the wine, such as 2-methyl butanoic acid and 3-methyl butanol, are produced by microorganisms isolated in the vineyards, as Gram-positive bacterium Paenibacillus, or the basidiomycetous fungus Sporobolomyces or the ascomycetous Aureobasidium. Therefore, there could be a relationship between some of the microbial species found in grapes and some detected aromas in wine, coming from the must of course (Verginer et al 2010).
Metagenomics as analytical tool of microbiota from grapes
Since conventional methods of isolation and cultivation of microorganisms are slow, laborious and some microbes cannot be grown up in the usual isolation media, massive sequencing methods or metagenomics are currently used. These consist of analysing all the DNA of a sample, and deducing which are the present microorganisms by comparing the sequences found with those of the databases. For bacteria the amplified DNA of V4 fragment from 16S RNA gene is used (Caporaso et al 2012).
This technique has been used with samples of botrytized wines (Bokulich et al 2012) and various LAB have been found (but not Oenococcus), including some not normally associated with wine. It has also been used to see the resident microbiota in wineries and how it changes with the seasons, resulting that in the surfaces of tanks and machinery of the cellar there is a majority of microorganisms neither related with wine nor harmful (Bokulich et al 2013).
With this technique Bokulich et al (2014) have also analysed the grapes and they have seen clear differences between the proportions of bacterial groups (and fungi) from different places, different varieties, as well as environmental or bio geographical conditions. For example, when analysing 273 samples of grape musts from California, the 3 varieties (Cabernet, Chardonnay and Zinfandel) are quite discriminated in a principal components analysis with respect to the bacterial communities found in each sample (Figure 2).
Thus, the dominant bacterial taxa or groups in a variety or given environment could provide some specifics traits on those wines, and this could explain some regional or terroir patterns in the organoleptic properties of these wines (Bokulich et al 2014).
Figure 2. Principal component analysis of bacterial communities of grape musts samples of Sonoma (California) from 3 varieties (Cabernet in red, Chardonnay in green and Zinfandel in blue) (Bokulich et al 2014).
We have also carried out a massive sequencing study with the same grape samples from which we have obtained isolates of O. oeni, as said before (Franquès et al 2015), and in more than 600,000 analysed sequences of 16S rRNA, we have found mainly Proteobacteria and Firmicutes. Among these gram-positive, we have found sequences of lactic acid bacteria (15%) and from these we have successfully confirmed the presence of O. oeni in 5% of the sequences. Therefore, we have isolated O. oeni from grapes and we have detected their DNA in the samples.
The bacterial microbiota of the vineyards and soil
As we see, microbiota of grapes and wine has been studied a little, but the soil microbiota has not been characterized. This one can define more clearly the terroir, which is influenced by the local climate and characteristics of the vineyard.
In Figure 3 the main genera found in different parts of the vine and soil are summarized (Gilbert et al 2014).
Figure 3. Main bacteria and fungi associated with organs and soil of Vitis vinifera (Gilbert et al 2014)
Recently an interesting scientific work (Zarraonaindia et al 2015) has been published on this subject, with the aim to see if the soil could be the main original source of bacteria that colonize the grapes. These authors took samples of soil, roots, leaves, flowers and grapes from Merlot vines, from different areas and years, of Suffolk, New York, and they analysed the bacterial DNA by 16S rRNA sequencing. They found that 40% of the species found were present in all samples of soil and roots, while there was more variability in leaves and fruits, and moreover, 40% of those found in leaves and fruits were also found in soils. All this suggests that many bacteria originate in the soil.
Regarding the type of bacteria, they found that Proteobacteria (especially Pseudomonas and Methylobacterium) predominated (Figure 4), mainly in the aerial parts of the plant. There were also Firmicutes as expected, and Acidobacteria and Bacteroides.
Figure 4. Composition of the bacterial community, at Phylum level, in samples from different organs of the vine and its soil (Zarraonaindia et al 2015).
Although variations were observed in all samples depending on the year (there may be different climatic conditions) and according to different edaphic factors (pH, C: N, humidity), the principal-components analysis (Figure 5) showed that the main types of samples (soil, roots, leaves, grapes) differ quite well, and bacterial taxon composition in samples of grape juice before fermentation is similar to that of grapes.
Figure 5. Principal-components analysis showing the similarities in terms of the composition of bacterial taxonomic groups, among sample types, including musts (Zarraonaindia et al 2015).
This suggests that the bacterial community found in grapes remains relatively stable until the processing to musts, and that it is more stable than the differences between organs. At the same time, a large number of representatives of bacterial phyla of the grapes come from the soil. This can be explained because when grapes are harvested by hand, they are often placed in boxes that are left on the ground, or for mechanical harvest, the machinery used removes the soil and generates dust, which can colonize the grapes.
Therefore, the soil microbiota is a source of bacteria associated with vines and may play a role in the must and therefore in the wine, and potentially in the formation of the terroir characteristics. Some of these bacteria may have some roles not yet known in productivity or disease resistance of the plant, or contribute to the organoleptic characteristics of wine (Zarraonaindia et al 2015).
In addition, and thinking in wine microorganisms responsible for fermentations, as said, in our laboratory we have confirmed that there are some O. oeni strains in grapes and we have confirmed this by detecting their DNA in the same grapes.
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