Category Archives: Microbiota

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

 

Human skin microbiota partly shared with our dog

December 25th, 2015

 

Diversity of the human microbiota in different parts of the body and between individuals

As I have commented in previous posts of this blog (Good Clostridia in our gut March 21st, 2015; Bacteria controlling what we eat October 12th, 2014; Bacteria of breast milk February 3rd, 2013), it becomes increasingly clear the importance of our microbiota, id est, all the micro-organisms, especially bacteria, with which we live.

The human microbiota varies from one individual to another, in relation to diet, age and the own genetic and phenotypic characteristics. Moreover, since we do not live isolated, there is also the influence of the environment, and of other people with we live, including our pets, dogs and others. They all have also their own microbiota.

The human body is home to many different microorganisms: bacteria (and archaea), fungi and viruses, that live on the skin, in the gut and in several other places in the body (Figure 1). While many of these microbes are beneficial to their human host, we know little about most of them. Early research focused on the comparison of the microorganisms found in healthy individuals with those found in people suffering from a particular disease. More recently, researchers have been interested in the more general issues, such as understanding how the microbiota is established and knowing the causes of the similarities and differences between the microbiota of different individuals.

Fig 1 Marsland

Figure 1. Types of microorganisms that live in different parts of the human body: bacteria (large circles), fungi (small circles right) and viruses (small circles left) (Marsland & Gollwitzer 2014)

 

Now we know that communities of microorganisms that are found in the gut of genetically related people tend to be more similar than those of people who are not related. Moreover, microbial communities found in the gut of unrelated adults living in the same household are more similar than those of unrelated adults living in different households (Yatsunenko et al 2012). However, these studies have focused on the intestine, and little is known about the effect of the relationship, cohabitation and age in microbiota of other parts of the body, such as skin.

 

Human skin microbiota

The skin is an ecosystem of about 1.8 m2 of various habitats, with folds, invaginations and specialized niches that hold many types of microorganisms. The main function of the skin is to act as a physical barrier, protecting the body from potential attacks by foreign organisms or toxic substances. Being also the interface with the external environment, skin is colonized by microorganisms, including bacteria, fungi, viruses and mites (Figure 2). On its surface there are proteobacteria, propionibacteria, staphylococci and some fungi such as Malassezia (an unicellular basidiomycetous). Mites such as Demodex folliculorum live around the hair follicles. Many of these microorganisms are harmless and often they provide vital functions that the human genome has not acquired by evolution. The symbiotic microorganisms protect human from other pathogenic or harmful microbes. (Grice & Segre 2011).

Fig 2 Grice

Figure 2. Schematic cross section of human skin with the different microorganisms (Grice & Segre 2011).

 

According to the commented diversity of microbiota, this is also very different depending on the region of skin (Figure 3), and therefore depending on the different microenvironments, that can be of three different characteristics: sebaceous or oily, wet and dry.

 

Fig 3 Grice

Figure 3. Topographic distribution of bacterial types in different parts of the skin (Grice & Segre 2011)

 

The skin is a complex network (structural, hormonal, nervous, immune and microbial) and in this sense it has been proven that the resident microbiota collaborates with the immune system, especially in the repair of wounds (Figure 4). As we see, particularly the lipopotheicoic acid (LTA), compound of the bacterial cell wall, can be released by Staphylococcus epidermidis and stimulates Toll-like receptors TLR2, which induce the production of antimicrobial peptides, and also stimulate epidermal keratinocytes via TLR3, which trigger the inflammation with production of interleukin and attracting leukocytes (Heath & Carbone 2013). All this to ensure the homeostatic protection and the defence against the potential pathogens. More information in the review of Belkaid & Segre (2014).

 

Fig 4 Heath Fig1 ni.2680-F1

Figure 4. Contribution of the resident microbiota to the immunity and wound repair (Heath & Carbone 2013)

At home we share microbiota, and with the dog

As mentioned earlier, environment influences the microbiota of an individual, and therefore, individuals who live together tend to share some of the microbiota. Indeed, it was recently studied by Song et al (2013), with 159 people and 36 dogs from 60 families (couples with children and / or dogs). They study the microbiota of gut, tongue and skin. DNA was extracted from a total of 1076 samples, amplifying the V2 region of the 16S rRNA gene with specific primers, and then it was proceeded to multiplex sequencing of high performance (High-Throughput Sequencing) with an Illumina GA IIx equipment. Some 58 million sequences were obtained, with an average of 54,000 per sample, and they were analysed comparing with databases to find out what kind of bacteria and in what proportions.

The results were that the microbial communities were more similar to each other in individuals who live together, especially for the skin, rather than the bowel or the tongue. This was true for all comparisons, including pairs of human and dog-human pairs. As shown in Figure 5, the number of bacterial types shared between different parts was greater (front, palms and finger pulps dog) of the skin of humans and their own dog (blue bars) than the human with dogs of other families (red bars), or dogs with people without dogs (green bars). We also see that the number of shared bacterial types is much lower when compared faecal samples or the tongue (Song et al 2013).

Fig 5 Song

Figure 5. Numbers of bacterial phylotypes (phylogenetic types) shared between adults and their dogs (blue), adults with other dogs (red) and adults who do not have dogs with dogs. There are compared (dog-human) fronts, hands, legs pulps, and also faecal samples (stool) and tongues. Significance of being different: *p<0.05, **p<0.001 (Song et al 2013)

 

This suggests that humans probably take a lot of microorganisms on the skin by direct contact with the environment and that humans tend to share more microbes with individuals who are in frequent contact, including their pets. Song et al. (2013) also found that, unlike what happens in the gut, microbial communities in the skin and tongue of infants and children were relatively similar to those of adults. Overall, these findings suggest that microbial communities found in the intestine change with age in a way that differs significantly from those found in the skin and tongue.

Although is not the main reason for this post, briefly I can say that the overall intestinal microbiota of dogs is not very different from humans in numbers (1011 per gram) and diversity, although with a higher proportion of Gram-positive (approx. 60% clostridial, 12% lactobacilli, 3% bifidobacteria and 3% corynebacteria) in dogs, and less Gram-negative (2% Bacteroides, 2% proteobacteria) (García-Mazcorro Minamoto & 2013).

 

Less asthma in children living with dogs

Although the relationship with the microbiota has not fully been demonstrated, some evidence of the benefits of having a dog has been shown recently, and for the physical aspects, not just for the psychological ones. Swedish researchers (Fall et al 2015) have carried out a study of all new-borns (1 million) in Sweden since 2001 until 2010, counting those suffering asthma at age 6. As the Swedes also have registered all dogs since 2001, these researchers were able to link the presence of dogs at home during the first year of the baby with the onset of asthma or no in children, and have come to the conclusion that children have a lower risk of asthma (50% less) if they have grown in the presence of a dog.

Similar results were obtained for children raised on farms or in rural environments, and thus having contact with other animals. All this would agree with the “hygiene hypothesis”, according to which the lower incidence of infections in Western countries, especially in urban people, would be the cause for increased allergic and autoimmune diseases (Okada et al 2010). In line with the hypothesis, it is believed that the human immune system benefits from living with dogs or other animals due to the sharing of the microbiota. However, in these Swede children living with dogs and having less risk of asthma there was detected a slight risk of pneumococcal disease. This links to the aforementioned hypothesis: more infections and fewer allergies (Steward 2015), but with the advantage that infections are easily treated or prevented with vaccines.

Fig 0 stray-dog-saves-baby

References

Belkaid Y, Segre JA (2014) Dialogue between skin microbiota and immunity. Science 346, 954-959

Fall T, Lundholm C, Örtqvist AK, Fall K, Fang F, Hedhammar Å, et al (2015) Early Exposure to Dogs and Farm Animals and the Risk of Childhood Asthma. JAMA Pediatrics 69(11), e153219

García-Mazcorro JF, Minamoto Y (2013) Gastrointestinal microorganisms in cats and dogs: a brief review. Arch Med Vet 45, 111-124

Heath WR, Carbone FR (2013) The skin-resident and migratory immune system in steady state and memory: innate lymphocytes, dendritic cells and T cells. Nature Immunology 14, 978-985

Marsland BJ, Gollwitzer ES (2014) Host–microorganism interactions in lung diseases. Nature Reviews Immunology 14, 827-835

Okada H, Kuhn C, Feillet H, Bach JF (2010) The “hygiene hypothesis” for autoimmune and allergic diseases: an update. Clin Exp Immunol 160, 1-9

Song SJ, Lauber C, Costello EK, Lozupone, Humphrey G, Berg-Lyons D, et al (2013) Cohabiting family members share microbiota with one another and with their dogs. eLife 2, e00458, 1-22

Steward D (2015) Dogs, microbiomes, and asthma risk: do babies need a pet ? MD Magazine, Nov 03

Yatsunenko T, Rey FE, Manary MJ, Trehan I, Dominguez-Bello MG, Contreras M, et al. 2012. Human gut microbiome viewed across age and geography. Nature 486, 222–7

 

 

The giant panda is herbivore but has the gut microbiota of a carnivore

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).

Fig0 panda bamboo

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).

Fig1 panda's thumb

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).

Fig2 Zhao F1 large

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.

Fig3 Ley

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.

Fig4 Zhu 2011-Fig1C

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).

Fig5 Xue F1 large

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).

Fig6 Xue Fig4

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).

References

Dierenfield ES, Hintz HF, Robertson JB, Van Soest PJ, Oftedal OT (1982) Utilization of bamboo by the giant panda. J Nutr 112, 636-641

Herron JC, Freeman S (2014) Evolutionary Analysis, 5th ed. Benjamin Cummings

Jin K, Xue C, Wu X, Qian J, Zhu Y et al. (2011) Why Does the Giant Panda Eat Bamboo? A Comparative Analysis of Appetite-Reward-Related Genes among Mammals. PLos One 6, e22602

Ley RE, Hamady M, Lozupone C, Turnbaugh PJ, Ramey RR et al. (2008) Evolution of Mammals and Their Gut Microbes. Science 320, 1647-1651

Li R, Fan W, Tian G, Zhu H, He L et 117 al. (2010) The sequence and de novo assembly of the giant panda genome. Nature 463, 311–317

Nie Y, Zhang Z, Raubenheimer D, Elser JJ, Wei W, Wei F (2015) Obligate herbivory in an ancestrally carnivorous lineage: the giant panda and bamboo from the perspective of nutritional geometry. Functional Ecology 29, 26–34

Rosen M (2015) Pandas’ gut bacteria resemble carnivores. Science News 19/05/2015

Wei G, Lu H, Zhou Z, Xie H, Wang A, Nelson K, Zhao L (2007) The microbial community in the feces of the giant panda (Ailuropoda melanoleuca) as determined by PCR-TGGE profiling and clone library analysis. Microb Ecol 54, 194–202

Wei F, Hu Y, Yan L, Nie Y, Wu Q, Zhang Z (2014) Giant Pandas Are Not an Evolutionary cul-de-sac: Evidence from Multidisciplinary Research. Mol Biol Evol 32, 4-12

Williams CL, Willard S, Kouba A, Sparks D, Holmes W et al. (2013) Dietary shifts affect the gastrointestinal microflora of the giant panda (Ailuropoda melanoleuca). J Anim Physiol Anim Nutr 97, 577-585

Xue Z, Zhang W, Wang L, Hou R, Zhang M et al. (2015) The bamboo-eating giant panda harbors a carnivore-like gut microbiota, with excessive seasonal variations. mBio 6(3), e00022-15

Zhao H, Yang JR, Xu H, Zhang J (2010) Pseudogenization of the Umami Taste Receptor Gene Tas1r1 in the Giant Panda Coincided with its Dietary Switch to Bamboo. Mol Biol Evol 27(12), 2669–2673

Zhu LF, Wu Q, Dai JY, Zhang SN, Wei FW (2011) Evidence of cellulose metabolism by the giant panda gut microbiome. Proc Natl Acad Sci USA 108, 17714–17719.

Bacteria of vineyard and terroir, and presence of Oenococcus in Priorat (South Catalonia) grapes

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.

Fig 1 garna-cari Priorat

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).

Fig 2 ACP Bokulich 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).

Fig 3 Gilbert 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.

Fig 4 microbiota vineyard

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.

Fig 5 distribució grups mostres OTUs

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.

References

Bae S, Fleet GH, Heard GM (2006) Lactic acid bacteria associated with wine grapes from several Australian vineyards. J Appl Microbiol 100, 712-727

Barata A, Malfeito-Ferreira M, Loureiro V (2012) The microbial ecology of wine grapes (Review). Int J Food Microbiol 153, 243-259

Bokulich NA, Joseph CML, Allen G, Benson AK, Mills DA (2012) Next-generation sequencing reveals significant bacterial diversity of botrytized wine. Plos One 7, e36357

Bokulich NA, Ohta M, Richardson PM, Mills DA (2013) Monitoring seasonal changes in winery-resident microbiota. Plos One 8, e66437

Bokulich NA, Thorngate JH, Richardson PM, Mills DA (2014) Microbial biogeography of wine grapes is conditioned by cultivar, vintage, and climate. PNAS nov 25, E139-E148

Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N, Owens SM, Betley J, Fraser L, Bauer M, Gormley N, Gilbert JA, Smith G, Knight R (2012) Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6, 1621–1624

Franquès J, Araque I, Portillo C, Reguant C, Bordons A (2015) Presence of autochthonous Oenococcus oeni in grapes and wines of Priorat in South Catalonia. Article in elaboration.

Garijo P, López R, Santamaría P, Ocón E, Olarte C, Sanz S, Gutiérrez AR (2011) Eur Food Res Technol 233, 359-365

Gilbert JA, van der Lelie D, Zarraonaindia I (2014) Microbial terroir for wine grapes. PNAS 111, 5-6

Renouf V, Claisse O, Lonvaud-Funel A (2005) Understanding the microbial ecosystem on the grape berry surface through numeration and identification of yeast and bacteria. Aust J Grape Wine Res 11, 316-327

Renouf V, Claisse O, Lonvaud-Funel A (2007) Inventory and monitoring of wine microbial consortia. Appl Microbiol Biotechnol 75, 149-164

Sieiro C, Cansado J, Agrelo D, Velázquez JB, Villa TG (1990) Isolation and enological characterization of malolactic bacteria from the vineyards of North-western Spain. Appl Environ Microbiol 56, 2936-2938

Verginer M, Leitner E, Berg G (2010) Production pf volatile metabolites by grape-associated microorganisms. J Agric Food Chem 58, 8344-8350

Zarraonaindia I, Owens SM, Weisenhorn P, West K, Hampton-Marcell J, Lax S, Bokulich NA, Mills DA, Martin G, Taghavi S, Van der Lelie D, Gilbert JA (2015) The soil microbiome influences grapevine-associated microbiota. mBio 6, e02527-14

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

The good bacteria of breast milk

Breast milk, besides being very nutritious, provides bioactive constituents that favor the development of the infant immune system and prevent diseases. From this point of view, the best known compounds are maternal immunoglobulins, immunocompetent cells and various antimicrobials. It also contains prebiotic substances, ie, several molecules such as oligosaccharides, which stimulate the growth of specific bacteria in the gut of the child.

However, other important constituents of breast milk, unsuspected until few years ago, are the bacteria. In fact, milk is not sterile, it contains microorganisms, primarily beneficial bacteria that help to establish the intestinal microbiota of the newborn, and which are the first to settle there. Although artificial milk are made to resemble the breast milk, they remain distinct and do not contain bacteria. And for this reason, the intestinal microbiota of breast-fed infants is different than those fed with artificial breast milk.

 

1-BL-mamant

Lactobacilli (image from AJC1Flickr) and suckling baby (© Photos.com)

Just a few weeks ago was published a work ( Cabrera-Rubio et al., 2012 ) in the American Journal of Clinical Nutrition that had a good coverage in media, blogs and networks ( click here for an example), because it shows the great diversity of bacteria present in the breast milk.

Although this work done by Valencian researchers (Cavanilles Institute, University of Valencia and CSIC-IATA) with Finnish researchers is not the first study that examines this issue, this study shows that bacteria are from very diverse species.

One of the novelties of this paper is the method used, taking advantage of the latest molecular biology: they studied the microbiome in breast milk, that is, the analysis of all possible bacteria present in the samples, by DNA sequencing, without the traditional isolation of living bacteria in plates. To do so, from the aseptically collected milk, DNA is extracted and the gene fragments of bacterial 16S rRNA are amplified by PCR. These amplified genes are sequenced by pyrosequencing (454 Roche GS-FLX), the most innovative and rapid sequencing technology: a machine of this allows about 400 million base pairs (bp) of DNA in 10 hours. From the rRNA gene of each possible bacteria some 500 bp are sequenced. Thus, in this study about 120,000 sequences have been analyzed, corresponding to 2600 sequences per milk sample.

By comparing these sequences with the databases and applying statistical methods conclusions can be drawn on what taxonomic groups (genera and species) bacteria are present and in what proportion.

 

2-Cabrera2012 generes bacteris

Predominant genera of bacteria in breast milk (Cabrera-Rubio et al., 2012)

As shown in the figure above, Cabrera et al. found in the milk of healthy mothers that the predominant genera are Leuconostoc, Weissella, Lactococcus and Staphylococcus, of which the first three are lactic acid bacteria. Although these are predominant in colostrum and milk during the first months, then other bacteria are increasing their numbers, such as Veillonella Leptotrichia (anaerobic gram-negative bacteria), which are typical commensal of the oral cavity. In total, about 1000 species have been found, that vary depending on the mother. Curiously, there are significant variations on whether delivery had been vaginal or cesarean, and on the obesity of the mother. The reasons for this are not yet clear.

And where the bacteria in breast milk come from ?

Besides the identifications made in this study of Cabrera et al. (2012) on the basis of DNA present, it has been observed by making viable counts that the total number of bacteria in breast milk is between 2·104 and 3·105 per ml (Juan Miguel Rodríguez), that is, a quantity not negligible . What is its origin?

The study of the microbiome of Cabrera et al. also concluded that the composition of different bacteria is somewhat different from that of other bacterial communities in the human body (the human bacterial niches: skin, mouth, digestive system, vagina, etc), and therefore the milk microbiome is not a particular subset of one of these niches.

The group Probilac from Universidad Complutense de Madrid,  whose head is Juan Miguel Rodriguez, a friend and colleague of Red BAL (Spanish network of lactic acid bacteria) is working in this area for years (ex: Martin et al 2003 , Martin et al 2004).

As discussed in a recent review published by this group (Fernández et al 2012), the bacteria present in the breast milk would come from three possible sources (figure below): skin bacteria from the same breast, the oral cavity of the infant, and the most surprising, commensal bacteria of the maternal gut that pass to milk by the entero-mammary pathway.

 

3-fig Fdez Review

Potential sources of bacteria present in human colostrum and milk, including the transit of intestinal commensal bacteria to the milk by the entero-mammary pathway (Fernández et al., 2012). DC: dendritic cells.

Indeed, several studies had shown that dendritic cells cross the intestinal epithelium (between enterocytes) and may take commensal bacteria of the gut lumen, incorporating them by endocytosis, but keeping them alive. See details in the following diagram.

 

4-JM Rodríguez dendritic LAB no lege

Dendritic cell capturing gut bacteria (Scheme of J.M. Rodríguez, group Probilac, Univ. Complutense de Madrid).

These dendritic cells travel through the circulatory system, reaching the mammary glands, where it seems that include bacteria to milk. This is the the entero-mammary pathway.

In this breast microbiota, bacteria from breast skin and from oral cavity of the child also would be incorporated. Some of these bacteria the child’s oral cavity are actually related to those of its gastrointestinal tract. As the first bacteria inhabiting this tract are those of the vaginal microbiota during birth (and intestinal if delivery is cesarean), this would explain the phylogeny of certain bacteria in the milk of these microbiota.

In summary, we see as the “good” bacteria (lactic acid bacteria, but also bifidobacteria and other) from maternal gut, by different ways, arrive to breast milk, and the reach the child’s gut, developing there the child’s microbiota, and helping to complete the neonatal immune system.

Bibliography

Cabrera-Rubio R, MC Collado, K Laitinen, S Salminen, E Isolauri, A Mira (2012) The human milk microbiome changes over lactation and is shaped by maternal weight and mode of delivery. American J Clinical Nutrition 96, 544–51

Grupo Probilac (Juan Miguel Rodríguez Gómez) Microbiota de la leche humana en condiciones fisiológicas: http://www.ucm.es/info/probilac/microbiota2.htm, Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid

Fernández L, S Langa, V Martín, A Maldonado, E Jiménez, R Martín, JM Rodríguez (2012) The human milk microbiota: Origin and potential roles in health and disease. Pharmacological Research http://dx.doi.org/10.1016/j.phrs.2012.09.001

Hunt KM JA Foster, LJ Forney, UME Schütte, DL Beck, Z Abdo, LK Fox, JE Williams, MK McGuire, MA McGuire (2011) Characterization of the diversity and temporal stability of bacterial communities in human milk. PLoS ONE 6:e21313.

Martín R, S Langa, C Revriego, E Jiménez, ML Marín, J Xaus, L Fernández, JM Rodríguez (2003) Human milk is a source of lactic acid bacteria for the infant gut. J Ped. 143, 754-758.

Martín R, S Langa, C reviriego, E Jiménez, ML Marín, M Olivares, J Boza, J Jiménez, L fernández, J Xaus, JM Rodríguez (2004) The commensal microflora of human milk: new perspectives for food bacteriotherapy and probiotics. Trends Food Sci Technol 15:121–7.

Other references

Adlerberth I (2006) Reduced enterobacterial and increased staphylococcal colonization of the infantile bowel: an effect of hygienic lifestyle. Pediatric Res 59, 96-101.

Albesharata R et al (2011) Phenotypic and genotypic analyses of lactic acid bacteria in local fermented food, breast milk and faeces of mothers and their babies. Syst App Microb 34, 148–155

Domínguez-Bello MG et al (2010) Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc Natl Acad Sci USA;107:11971–5.

Huurre A et al (2008) Mode of delivery—effects on gut microbiota and humoral immunity. Neonatology;93:236–40

LeBouder E et al (2006) Modulation of neonatal microbial recognition: TLRmediated innate immune responses are specifically and differentially modulated by human milk. J Immunol;176:3742–52.

Martín R et al (2009) Isolation of bifidobacteria from breast milk and assessment of the bifidobacterial population by PCR-denaturing gradient gel electrophoresis and quantitative real-time PCR. Appl Environ Microbiol 75:965–9.

Pérez PF et al (2007) Bacterial imprinting of the neonatal immune system: lessons from maternal cells? Pediatrics 119: 724–732.

Rescigno M et al (2001) Dendritic cells shuttle microbes across gut epithelial monolayers. Immunobiology 204:572–81.

Stockinger S et al (2001) Establishment of intestinal homeostasis during the neonatal period. Cell Mol Life Sci;68: 3699–712.

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