12th August 2017
Probiotics are living microorganisms that, when ingested in adequate amounts, can have a positive effect on the health of guests (FAO / WHO 2006; World Gastroenterology Organization 2011, Fontana et al., 2013). Guests can be humans but also other animals. Lactic acid bacteria, especially the genus Lactobacillus and Bifidobacterium, both considered as GRAS (Generally recognized as safe), are the microbes most commonly used as probiotics, but other bacteria and some yeasts can also be useful. Apart from being able to be administered as medications, probiotics are commonly consumed for millennia as part of fermented foods, such as yoghurt and other dairy products (see my article “European cheese from 7400 years ago..” “December 26th, 2012). As medications, probiotics are generally sold without prescription, over-the-counter (OTC) in pharmacies.
I have already commented on the other posts of this blog the relevance of probiotics (“A new probiotic modulates microbiota against hepatocellular carcinoma” August 24th, 2016), as well as the microbiota that coexists with our body (“Bacteria in the gut controlling what we eat” October 12th, 2014; “The good bacteria of breast milk” February 3rd, 2013) and other animals (“Human skin microbiota … and our dog” December 25th, 2015; “The herbivore giant panda …. and its carnivore microbiota” September 30th, 2015).
Besides lactic acid bacteria and bifidobacteria, other microorganisms that are also used to a certain extent as probiotics are the yeast Saccharomyces cerevisiae, some strains of Escherichia coli, and some Bacillus, as we will see. Some clostridia are also used, related to what I commented in a previous post of this blog by March 21st, 2015 (“We have good clostridia in the gut ...”).
In fact, Bacillus and clostridia have in common the ability to form endospores. And both groups are gram-positive bacteria, within the taxonomic phylum Firmicutes (Figure 1), which also includes lactic acid bacteria. However, bacilli (Bacillus and similar ones, but also Staphylococcus and Listeria) are more evolutionarily closer to lactobacillalles (lactic acid bacteria) than to clostridia ones. The main physiological difference between Clostridium and Bacillus is that the first are strict anaerobes while Bacillus are aerobic or facultative anaerobic.
Figure 1. Phylogenetic tree diagram of Gram-positive bacteria (Firmicutes and Actinobacteria). Own elaboration.
Bacterial endospores (Figure 2) are the most resistant biological structures, as they survive extreme harsh environments, such as UV and gamma radiation, dryness, lysozyme, high temperatures (they are the reference for thermal sterilization calculations), lack of nutrients and chemical disinfectants. They are found in the soil and in the water, where they can survive for very long periods of time.
Figure 2. Endospores (white parts) of Bacillus subtilis in formation (Image of Simon Cutting).
Bacillus in fermented foods, especially Asian
Several Bacillus are classically involved in food fermentation processes, especially due to their protease production capacity. During fermentation, this contributes to nutritional enrichment with amino acids resulting from enzymatic proteolysis.
Some of these foods are fermented rice flour noodles, typical of Thailand and Burma (nowadays officially Myanmar). It has been seen that a variety of microorganisms (lactic acid bacteria, yeasts and other fungi) are involved in this fermentation, but also aerobic bacteria such as B. subtilis. It has been found that their proteolytic activity digests and eliminates protein rice substrates that are allergenic, such as azocasein, and therefore they have a beneficial activity for the health of consumers (Phromraksa et al. 2009).
However, the best-known fermented foods with Bacillus are the alkaline fermented soybeans. As you know, soy (Glycine max) or soya beans are one of the most historically consumed nourishing vegetables, especially in Asian countries. From they are obtained “soy milk”, soybean meal, soybean oil, soybean concentrate, soy yogurt, tofu (soaked milk), and fermented products such as soy sauce, tempeh, miso and other ones. Most of them are made with the mushroom Rhizopus, whose growth is favoured by acidification or by direct inoculation of this fungus. On the other hand, if soy beans are left to ferment only with water, the predominant natural microbes fermenting soy are Bacillus, and in this way, among other things, the Korean “chongkukjang” is obtained, “Kinema” in India, the “thua nao” in northern Taiwan, the Chinese “douchi”, the “chine pepoke” from Burma, and the best known, the Japanese “natto” (Figure 3). Spontaneous fermentation with Bacillus gives ammonium as a by-product, and therefore is alkaline, which gives a smell not very good to many of these products. Nevertheless, natto is made with a selected strain of B. subtilis that gives a smoother and more pleasant smell (Chukeatirote 2015).
These foods are good from the nutritional point of view as they contain proteins, fibre, vitamins, and they are of vegetable or microbial origin. In addition, the advertising of the commercial natto emphasizes, besides being handmade and sold fresh (not frozen), its probiotic qualities, saying that B. subtilis (Figure 4) promotes health in gastrointestinal, immunologic, cardiovascular and osseous systems (www.nyrture.com). They say the taste and texture of natto are exquisite. It is eaten with rice or other ingredients and sauces, and also in the maki sushi. We must try it !
Figure 3. “Natto”, soybeans fermented with B. subtilis, in a typical Japanese breakfast with rice (Pinterest.com).
Figure 4. Coloured electronic micrograph of Bacillus subtilis (Nyrture.com).
Bacillus as probiotics
The endospores are the main advantage of Bacillus being used as probiotics, thanks to their thermal stability and to survive in the gastric conditions (Cutting 2011). Although Clostridium has also this advantage, its strict anaerobic condition makes its manipulation more complex, and moreover, for the “bad reputation” of this genus due to some well-known toxic species.
Unlike other probiotics such as Lactobacillus or Bifidobacterium, Bacillus endospores can be stored indefinitely without water. The commercial products are administered in doses of 10^9 spores per gram or per ml.
There are more and more commercial products of probiotics containing Bacillus, both for human consumption (Table 1) and for veterinary use (Table 2). In addition, there are also five specific products for aquaculture with several Bacillus, and also shrimp farms are often using products of human consumption (Cutting 2011).
For use in aquaculture, probiotic products of mixtures of Bacillus (B. thuringiensis, B. megaterium, B. polymixa, B. licheniformis and B. subtilis) have been obtained by isolating them from the bowel of the prawn Penaeus monodon infected with vibriosis. They have been selected based on nutrient biodegradation and the inhibitory capacity against the pathogen Vibrio harveyi (Vaseeharan & Ramasamy 2003). They are prepared freeze-dried or microencapsulated in sodium alginate, and it has been shown to significantly improve the growth and survival of shrimp (Nimrat et al., 2012).
As we see for human consumption products, almost half of the brands (10 of 25) are made in Vietnam. The use of probiotic Bacillus in this country is more developed than in any other, but the reasons are not clear. Curiously, as in other countries in Southeast Asia, there is no concept of dietary supplements and probiotics such as Bacillus are only sold as medications approved by the Ministry of Health. They are prescribed for rotavirus infection (childhood diarrhoea) or immune stimulation against poisoning, or are very commonly used as a therapy against enteric infections. However, it is not clear that clinical trials have been carried out, and they are easy-to-buy products (Cutting 2011).
Table 1. Commercial products of probiotics with Bacillus, for human consumption (modified from Cutting 2011).
|Product||Country where it is made||Species of Bacillus|
|Bactisubtil ®||France||B. cereus|
|Bibactyl ®||Vietnam||B. subtilis|
|Bidisubtilis ®||Vietnam||B. cereus|
|Bio-Acimin ®||Vietnam||B. cereus and 2 other|
|Biobaby ®||Vietnam||B. subtilis and 2 other|
|Bio-Kult ®||United Kingdom||B. subtilis and 13 other|
|Biosporin ®||Ukraine||B. subtilis + B. licheniformis|
|Biosubtyl ®||Vietnam||B. cereus|
|Biosubtyl DL ®||Vietnam||B. subtilis and 1 other|
|Biosubtyl I and II ®||Vietnam||B. pumilus|
|Biovicerin ®||Brazil||B. cereus|
|Bispan ®||South Korea||B. polyfermenticus|
|Domuvar ®||Italy||B. clausii|
|Enterogermina ®||Italy||B. clausii|
|Flora-Balance ®||United States||B. laterosporus *|
|Ildong Biovita ®||Vietnam||B. subtilis and 2 other|
|Lactipan Plus ®||Italy||B. subtilis *|
|Lactospore ®||United States||B. coagulans *|
|Medilac-Vita ®||China||B. subtilis|
|Nature’s First Food ®||United States||42 strains, including 4 B.|
|Neolactoflorene ®||Italy||B. coagulans * and 2 other|
|Pastylbio ®||Vietnam||B. subtilis|
|Primal Defense ®||United States||B. subtilis|
|Subtyl ®||Vietnam||B. cereus|
|Sustenex ®||United States||B. coagulans|
* Some labelled as Lactobacillus or other bacteria are really Bacillus
Table 2. Commercial products of probiotics with Bacillus, for veterinary use (modified from Cutting 2011).
|Product||Animal||Country where it is made||Species of Bacillus|
|AlCare ®||Swine||Australia||B. licheniformis|
|BioGrow ®||Poultry, calves and swine||United Kingdom||B. licheniformis and B. subtilis|
|BioPlus 2B ®||Piglets, chickens, turkeys||Denmark||B. licheniformis and B. subtilis|
|Esporafeed Plus ®||Swine||Spain||B. cereus|
|Lactopure ®||Poultry, calves and swine||India||B. coagulans *|
|Neoferm BS 10 ®||Poultry, calves and swine||France||B. clausii|
|Toyocerin ®||Poultry, calves, rabbits and swine||Japan||B. cereus|
The Bacillus species that we see in these Tables are those that really are found, once the identification is made, since many of these products are poorly labelled as Bacillus subtilis or even as Lactobacillus (Green et al. 1999; Hoa et al. 2000). These labelling errors can be troubling for the consumer, and especially for security issues, since some of the strains found are Bacillus cereus, which has been shown to be related with gastrointestinal infections, since some of them produce enterotoxins (Granum & Lund 1997; Hong et al. 2005)
The probiotic Bacillus have been isolated from various origins. For example, some B. subtilis have been isolated from the aforementioned Korean chongkukjang, which have good characteristics of resistance to the gastrointestinal tract (GI) conditions and they have antimicrobial activity against Listeria, Staphylococcus, Escherichia and even against B. cereus (Lee et al. 2017).
One of the more known probiotics pharmaceuticals is Enterogermina ® (Figure 5), with B. subtilis spores, which is recommended for the treatment of intestinal disorders associated with microbial alterations (Mazza 1994).
Figure 5. Enterogermina ® with spores of Bacillus subtilis (Cutting 2011)
Bacillus in the gastrointestinal tract: can they survive there ?
It has been discussed whether administered spores can germinate in the GI tract. Working with mice, Casula & Cutting (2002) have used modified B. subtilis, with a chimeric gene ftsH-lacZ, which is expressed only in vegetative cells, which can be detected by RT-PCR up to only 100 bacteria. In this way they have seen that the spores germinate in significant numbers in the jejunum and in the ileum. That is, spores could colonize the small intestine, albeit temporarily.
Similarly, Duc et al. (2004) have concluded that B. subtilis spores can germinate in the gut because after the oral treatment of mice, in the faeces are excreted more spores that the swallowed ones, a sign that they have been able to proliferate. They have also detected, through RT-PCR, mRNA of vegetative bacilli after spore administration, and in addition, it has been observed that the mouse generates an IgG response against bacterial vegetative cells. That is, spores would not be only temporary stagers, but they would germinate into vegetative cells, which would have an active interaction with the host cells or the microbiota, increasing the probiotic effect.
With all this, perhaps it would be necessary to consider many Bacillus as not allochthonous of the GI tract, but as bacteria with a bimodal growth and sporulation life cycle, both in the environment and in the GI tract of many animals (Hong et al. 2005).
Regarding the normal presence of Bacillus in the intestine, when the different microorganisms inhabiting the human GI tract are studied for metagenomic DNA analysis of the microbiota, the genus Bacillus does not appear (Xiao et al., 2015). As we can see (Figure 6), the most common are Bacteroides and Clostridium, followed by various enterobacteria and others, including bifidobacteria.
Figure 6. The 20 bacterial genera more abundant in the mice (left) and human (right) GI tract (Xiao et al. 2015).
In spite of this, several species of Bacillus have been isolated from the GI tract of chickens, treating faecal samples with heat and ethanol to select only the spores, followed by aerobic incubation (Barbosa et al. 2005). More specifically, the presence of B. subtilis in the human microbiota has been confirmed by selective isolation from biopsies of ileum and also from faecal samples (Hong et al. 2009). These strains of B. subtilis exhibited great diversity and had the ability to form biofilms, to sporulate in anaerobiosis and to secrete antimicrobials, thereby confirming the adaptation of these bacteria to the intestine. In this way, these bacteria can be considered intestinal commensals, and not only soil bacteria.
Security of Bacillus as probiotics
The oral consumption of important amounts of viable microorganisms that are not very usual in the GI treatment raises additional doubts about safety. Even more in the use of species that do not have a history of safe use in foods, as is the case of sporulated bacteria. Even normal bowel residents may sometimes act as opportunistic pathogens (Sanders et al. 2003).
With the exception of B. anthracis and B. cereus, the various species of Bacillus are generally not considered pathogenic. Of course, Bacillus spores are commonly consumed inadvertently with foods and in some fermented ones. Although Bacillus are recognized as GRAS for the production of enzymes, so far the FDA has not guaranteed the status of GRAS for any sporulated bacteria with application as a probiotic, neither Bacillus nor Clostridium. While Lactobacillus and Bifidobacterium have been the subject of numerous and rigorous tests of chronic and acute non-toxicity, and a lot of experts have reviewed data and have concluded that they are safe as probiotics, there is no toxicity data published on Bacillus in relation to their use as probiotics. When reviewing articles on Medline with the term “probiotic” and limited to clinical studies, 123 references appear, but Bacillus does not appear in any of them (Sanders et al. 2003).
Instead, there are some clinical studies where Bacillus strains have been detected as toxigenic. All this explains that some probiotic Bacillus producers refer to them with the misleading name of Lactobacillus sporogenes, a non-existent species, as can be seen from NCBI (https://www.ncbi.nlm.nih.gov/taxonomy/?term = Lactobacillus + sporogenes).
Finally, we should remember the joint report on probiotics of FAO (United Nations Food and Agriculture Organization) and WHO (World Health Organization) (FAO / WHO 2006), which suggests a set of Guidelines for a product to be used as a probiotic, alone or in the form of a new food supplement. These recommendations are:
- The microorganism should be well characterized at the species level, using phenotypic and genotypic methods (e.g. 16S rRNA).
- The strain in question should be deposited in an internationally recognized culture collection.
- To evaluate the strain in vitro to determine the absence of virulence factors: it should not be cytotoxic neither invades epithelial cells, and not produce enterotoxins or haemolysins or lecithinases.
- Determination of its antimicrobial activity, and the resistance profile, including the absence of resistance genes and the inability to transfer resistance factors.
- Preclinical evaluation of its safety in animal models.
- Confirmation in animals demonstrating its effectiveness.
- Human evaluation (Phase I) of its safety.
- Human evaluation (Phase II) of its effectiveness (if it does the expected effect) and efficiency (with minimal resources and minimum time).
- Correct labelling of the product, including genus and species, precise dosage and conservation conditions.
The use of Bacillus as probiotics, especially in the form of dietary supplements, is increasing very rapidly. More and more scientific studies show their benefits, such as immune stimulation, antimicrobial activities and exclusive competition. Their main advantage is that they can be produced easily and that the final product, the spores, is very stable, which can easily be incorporated into daily food. In addition, there are studies that suggest that these bacteria may multiply in GI treatment and may be considered as temporary stagers (Cutting 2011).
On the other hand, it is necessary to ask for greater rigor in the selection and control of the Bacillus used, since some, if not well identified, could be cause of intestinal disorders. In any case, since the number of products sold as probiotics that contain the sporulated Bacillus is increasing a lot, one must not assume that all are safe and they must be evaluated on a case-by-case basis (Hong et al. 2005).
Barbosa TM, Serra CR, La Ragione RM, Woodward MJ, Henriques AO (2005) Screening for Bacillus isolates in the broiler gastrointestinal tract. Appl Environ Microbiol 71, 968-978.
Casula G, Cutting SM (2002) Bacillus probiotics: Spore germination in the gastrointestinal tract. Appl Environ Microbiol 68, 2344-2352.
Chukeatirote E (2015) Thua nao: Thai fermented soybean. J Ethnic Foods 2, 115-118.
Cutting SM (2011) Bacillus probiotics. Food Microbiol 28, 214-220.
Duc LH, Hong HA, Barbosa TM, Henriques AO, Cutting SM (2004) Characterization of Bacillus probiotics available for human use. Appl Environ Microbiol 70, 2161-2171.
FAO/WHO (2006) Probiotics in food. Health and nutritional properties and guidelines for evaluation. Fao Food and Nutrition Paper 85. Reports of Joint FAO/WHO expert consultations.
Fontana L, Bermudez-Brito M, Plaza-Diaz J, Muñoz-Quezada S, Gil A (2013) Sources, isolation, characterization and evaluation of probiotics. Brit J Nutrition 109, S35-S50.
Granum, P. E., T. Lund (1997) Bacillus cereus and its food poisoning toxins. FEMS Microbiol. Lett. 157:223–228.
Green, D. H., P. R. Wakeley, A. Page, A. Barnes, L. Baccigalupi, E. Ricca, S. M. Cutting (1999) Characterization of two Bacillus probiotics. Appl Environ Microbiol 65, 4288–4291.
Hoa, N. T., L. Baccigalupi, A. Huxham, A. Smertenko, P. H. Van, S. Ammendola, E. Ricca, A. S. Cutting (2000) Characterization of Bacillus species used for oral bacteriotherapy and bacterioprophylaxis of gastrointestinal disorders. Appl Environ Microbiol 66, 5241–5247.
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Lee S, Lee J, Jin YI, Jeong JC, Hyuk YH, Lee Y, Jeong Y, Kim M (2017) Probiotic characteristics of Bacillus strains isolated from Korean traditional soy sauce. LWT – Food Sci Technol 79, 518-524.
Mazza P (1994) The use of Bacillus subtilis as an antidiarrhoeal microorganism. Boll Chim. Farm. 133, 3-18.
Nimrat S, Suksawat S, Boonthai T, Vuthiphandchai V (2012) Potential Bacillus probiotics enhance bacterial numbers, water quality and growth during early development of white shrimp (Litopenaeus vannamei). Veterinary Microbiol 159, 443-450.
Phromraksa P, Nagano H, Kanamaru Y, Izumi H, Yamada C, Khamboonruang C (2009) Characterization of Bacillus subtilis isolated from Asian fermented foods. Food Sci Technol Res 15, 659-666.
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Vaseeharan, B., P. Ramasamy (2003) Control of pathogenic Vibrio spp. by Bacillus subtilis BT23, a possible probiotic treatment for black tiger shrimp Penaeus monodon. Lett Appl Microbiol 36, 83–87
World Gastroenterology Organisation Global Guidelines (2011) Probiotics and Prebiotics.
Xiao et al. (2015) A catalogue of the mouse gut metagenome. Nature Biotechnol 33, 1103-1108.
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.
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).
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.
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).
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).
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.
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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
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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.
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.
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.
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.
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.
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).
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
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.
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.
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)
|GABA (gamma-amino-butyric acid)||Lactobacillus, Bifidobacterium|
|Norepinephrine||Escherichia, Bacillus, Saccharomyces|
|Serotonin||Candida, Streptococcus, Escherichia, Enterococcus|
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.
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
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.
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.
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.
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.
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.
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
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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.
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