Category Archives: Genetics and molecular biology
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.
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
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 is really surprising, but it seems so: Italian and Austrian researchers have published a paper (Campisano et al. 2014) which shows that the bacterial species Propionibacterium acnes, related to human acne, can be found as obligate endophytes in bark tissues of Vitis vinifera, the grapevine.
Some bacterial pathogens of humans, such as Salmonella, are able to colonize plant tissues but temporarily and opportunistically (Tyler & Triplett 2008). In fact, there is a temporary mutual benefit between plants and bacteria, so some of these enterobacteria pathogenic to plants do not live endophytically and can be beneficial for them. These pathogens to humans, in its life cycle, use plants as alternative hosts to survive the environment, passing to the plants through contaminated irrigation water. Therefore, some bacteria are often temporary endophyte guests of plants.
But on the other hand, there are relatively rare cases of bacteria changing the host and adapting to the new host, finally being endophytes. This horizontal transfer happens mostly between evolutionarily close hosts, such as symbiotic bacteria of aphids (insects), which has proven to transfer to other species of aphids (Russell & Moran 2005). It has also been suggested the horizontal transfer of beneficial lactic acid bacteria (Lactobacillus reuteri) in the intestinal tract of vertebrates, since strains of this L. reuteri are similar in several species of mammals and birds.
Well, going beyond, the work of Campisano et al. subject of this review, concludes that bacteria associated with human acne should have passed on the vine, that is, the bacteria would have made a horizontal transfer interregnum, from plants to mammals.
Propionibacterium acnes type Zappae
Acne, as you know, is a common human skin disease, consisting of an excess secretion of the pilosebaceous glands caused by hormonal changes, especially teenagers. The glands become inflamed, the pores obstructed and scarring appears. The microorganism associated with these infections is the opportunistic commensal bacterium P. acnes, a gram-positive anaerobic aero tolerant rod, which fed fatty acids produced by the glands.
Young with acne (Wikimedia, public)
Propionibacterium acnes at the scanning electron microscope (left) and dyed with violet crystal (right). From Abate ME (2013) Student Pulse 5, 9, 1-4.
Interestingly, other species of the same genus Propionibacterium well known in microbial biotechnology industry are used for the production of propionic acid, vitamin B12, and the Swiss cheeses Gruyere or Emmental.
Campisano et al. have made a study of the vineyard endomicrobioma by the sequencing technique (Roche 454) amplifying the V5-V9 hyper variable region of the bacterial 16S rDNA present in the tissues of vine. In 54 of the 60 plants analyzed, between 0.5% and 5% of the found sequences correspond to the species Propionibacterium acnes. This observation has been confirmed by fluorescent in situ hybridization (FISH) with fluorochromes and specific probes of P. acnes.
Location of P. acnes (fluorescent blue spots) in the bark of a vine stem, seen with FISH microscopy with specific probes for this bacterium (Campisano et al 2004).
The authors of this work proposed for this bacterium the name of P. acnes Zappae, in memory of the eccentric musician and composer Frank Zappa, to emphasize the unexpected and unconventional habitat of this type of P. acnes.
Frank Zappa (1940-1993), the eccentric and satiric singer, musician and composer. Photo: Frank Zappa reviews.
And how did this human bacteria arrive into the vineyard?
To solve this riddle, Campisano et al. have taken the 16S rDNA sequences and from other genes (recA and tly) from these strains of P. acnes Zappae found in vine and have compared with those P. acnes of human origin in databases. Comparing phylogenies and clusters deducted from them, these researchers have concluded that P. a. Zappae has diversified evolutionarily recently. Studying in detail the recA gene sequences of P. a. Zappae, and taking into account the likely mutation rate and generation time (about 5 hours), they deduce that the diversification from other P. acnes occurred 6000-7000 years ago.
This date coincides with the known domestication of the vine by humans, which is believed to have occurred about 7000 years ago in the southern Caucasus, between the Black Sea and the Caspian Sea, the area of modern Turkey, Georgia, Armenia and Iran (Berkowitz 1996). The vineyard has its origins in a wild subspecies of Vitis that survived the Ice Age and was domesticated. This plant came out to three subspecies, and one of them, Vitis vinifera pontica, spread in the mentioned area and further south in Mesopotamia and then to all south Europe thanks to the Phoenicians.
Therefore, the conclusion is that P. acnes Zappae originated from human P. acnes 7000 years ago, by contact of human hands with grapes and other parts of the vineyard during the harvest and carrying them. As the authors say, this case would be the first evidence of horizontal transfer interregnum, from humans to plants, of a obligate symbiotic bacterium. This also makes more remarkable the adaptability of bacteria. Their ability to exploit new habitats can have unforeseen impacts on the evolution of host-symbiont relationship or even host-pathogen.
Harvesting by hand in Chile (Fine Wine and Good Spirits)
Berkowitz M (1996) World’s earliest wine. Archaeology 49, 5, Sept./Oct.
Campisano Aet al. (2014) Interkingdom transfer of the acne-causing agent, Propionibacterium acnes, from human to grapevine. Mol Biol Evol 31, 1059-1065.
Gruber K (4 march 2014) How grapevines got acne bacteria. Nature News 4 march 2014.
Russell JA, NA Moran (2005) Horizontal transfer of bacterial symbionts: heritability and fitness effects in a novel aphid host. Appl Environ Microbiol 71, 7987-7994.
Tyler HL, EW Triplett (2008) Plants as a habitat for beneficial and/or human pathogenic bacteria. Ann Rev Phytopathol 46, 53-73.
Walter J, RA Britton, S Roos (2011) PNAS 108, 4645-4652.
Tuberculosis and its agent
Tuberculosis is a common and often deadly infectious disease if left untreated, caused by mycobacteria, mainly Mycobacterium tuberculosis. Mycobacteria such as M. tuberculosis and M. leprae (the leprosy) are considered as gram-positive bacteria because they have glycopeptides, but they are not stained with crystal violet, because outside of glycopeptide they have a good layer of fat, called the mycolic acid (Figure 1). Precisely the prefix “myco” comes from the fungic appearance on the surface of liquid cultures of these mycobacteria.
Figure 1. Mycobacterium tuberculosis with his particular cell wall, and the more used antibiotics against this bacterium. (Figure from National Institute of Allergy and Infectious Diseases)
This mycobacterium was discovered as causing tuberculosis by the German Robert Koch (1882) and for this reason, it is also called Koch’s bacillus. It is a strict aerobic bacterium, since it needs high levels of oxygen, and therefore mostly infects the lungs. It has a generation time of about 15-20 hours, so it has a very slow growing compared to other bacteria (remember the 20 minutes in Escherichia coli). From a taxonomic point of view they are Actinobacteria, or gram-positive of high G+ C, as corynebacteria or Actinomycetales.
The virulence of M. tuberculosis is very complex and has many facets (Todar K.). Although apparently it produces no toxin, it has a large repertoire of physiological and structural properties that explain its virulence and pathology of tuberculosis. Once in the lungs, the bacteria are captured by alveolar macrophages, but they cannot be digested due to the structure of the bacterial cell wall and because bacteria neutralize reactive compounds from macrophages. The mycolic acid also makes the cell not permeable to lysozyme. M. tuberculosis can grow intracellularly without being affected by the immune system, and at the same time, it secretes several proteins involved in the pathogenesis. For instance, the antigen 85 complex, which binds fibronectin, facilitates the formation of tubercles in the lungs (Figure 2).
Figure 2. Progress of infection by M. tuberculosis in the lungs, with the formation of tubercles (From humanorgans.org/tuberculosis)
Tuberculosis usually attacks the lungs but can also affect many other organs. The classic symptoms of tuberculosis are a chronic cough with bloody sputum, fever and other symptoms. The diagnosis relies on radiology of the thorax, a tuberculin skin test and blood analysis, and a microscopic examination and microbiological culture of body fluids. The treatment of tuberculosis is difficult and requires long treatment with various antibiotics. The most commonly used are rifampicin and isoniazid (Figure 1). However, antibiotic resistance is a growing problem in some types of tuberculosis. Prevention is based mainly on vaccination, usually with the Bacillus Calmette-Guérin vaccine (BCG).
Tuberculosis is transmitted by air when infected people cough, sneeze or spit. One third of the world’s current population is infected with M. tuberculosis, and there is a new infected person every second. However, in most of these cases the disease is not fully developed, as the latent and asymptomatic infections are the most common. Approximately one in ten latent infections eventually it progress to active disease, which, if left untreated, kills more than half of the victims. In one year (2004), the global statistics included 14.6 million chronic active cases, 8.9 million new cases and 1.6 million deaths, mostly in developing countries. In addition, a growing number of people in the developed world are contracting tuberculosis because their immune systems are compromised by immunosuppressive drugs, substance abuse, or AIDS.
When the human tuberculosis originated ?
The origin of the population infectious diseases (such as plague, cholera, etc.), id est., those that spread easily where there are many humans together, has been associated with the Neolithic demographic transition, about 10,000 years ago, when the revolution of agriculture allowed the establishment of the first sedentary villages, with numbers of humans living together. Many of these diseases are associated with other animals, from which they are transmitted to humans. Until recently it was believed that tuberculosis was of this type, having also led the Neolithic, but in fact the majority of tuberculosis have no relationship with other animals.
However, a recent study (Comas et al. 2013) provides data in the sense that the disease originated long before, more than 70,000 years ago, going with the modern humans out of Africa and spreading to the world. Effectively, this work of 22 co-authors from 9 countries (and the first author of which is the young Valencian Iñaki Comas) have analyzed and compared the genomes of 259 strains of the complex M. tuberculosis to see their evolutionary history. In doing so, within the 4.4 Mb genome of this species the authors have identified 34,167 SNPs polymorphic sites, i.e., sites of DNA that have some different nucleotide in function of strains, and with which they have been able to reconstruct the phylogeny of this bacterium. In this way, they have confirmed seven lineages (groups of strains) that had been suggested by other techniques. The most interesting thing is that this phylogeny of genomes of M. tuberculosis is very similar to the human mitochondrial genome phylogeny, as shown in Figure 3. Therefore, this suggests that the evolution of tuberculosis bacteria goes parallel to that of modern humans.
Figure 3. The phylogeny of genomes from 220 strains of M. tuberculosis, with the different lineages (MTBC, left), is similar to that of mitochondrial genomes (right) of 4995 humans, with their main haplogroups (from Comas et al. 2013, Fig. 1 c, d).
Based on these phylogenies and on the frequencies of mutations observed in the genomes of M. tuberculosis, the origin of the different lineages of this bacterium has been established, calculating at what time of the human Palaeolithic the different branches were appearing, from approx. 73,000 to 42,000 years (Figure 4), which, having seen the parallel with mitochondrial DNA, coincide with the proposed dates of the modern humans expansion from Africa to Eurasia.
Figure 4. Expansion of complex M. tuberculosis going out of Africa, with the origin of different lineages and the approximate data (thousands years) of the evolutionary branches (from Comas et al. 2013, Fig. 2a).
Therefore, we can conclude that tuberculosis and its bacterial agent has been a constant companion of the modern humans during their evolution and their global spread, at least in the last 70,000 years, and also that the bacterium M. tuberculosis has been able to adapt to changes in human population. In addition, in this regard last years an increase in resistant strains to multiple antibiotics has been observed. The study of these mechanisms of bacterial adaptation may help to predict future patterns of disease and to design rational strategies to fight it.
Comas, Iñaki, et al. (2013) Out-of-Africa migration and Neolithic coexpansion of Mycobacterium tuberculosis with modern humans. Nature Genetics 45, 1176-1182.
Todar K., Todar’s Online Textbook of Bacteriology.
Mycobacterium tuberculosis in wikipedia: http://en.wikipedia.org/wiki/Mycobacterium_tuberculosis
Tuberculosis in viquipèdia (Catalan): http://ca.wikipedia.org/wiki/Tuberculosi.
and use the translator buttons with flags, at right corner.
Thanks for your collaboration
The spotted hyena (Crocuta crocuta), also known as laughing hyena, is the best known and greatest species of hyena, living in Sub-Saharan Africa. Although not considered in immediate danger of extinction, their numbers have been increasingly shrinking, like all other large African mammals and their total number is estimated at about 40,000. Most of them live in national parks of the East Africa, especially in the Serengeti in Tanzania. In the rest of western and southern Africa, populations in many cases are lower than 1000 individuals in each country, and isolated from each other, so in real danger of extinction.
The spotted hyena (Crocuta crocuta). Photo: Tophat21 (animalswikia.com)
It is the carnivorous mammal with more complexity of social behaviour, similar to the cercopithecine primates (baboons and macaques), and because of this, his intelligence is comparable to those primates and in some respects even to the chimpanzees.
They live in communities, clans, of about 40 to 80 individuals and these societies are matriarchal: females, larger than males, are dominant, with even the lowest ranking females being dominant over the highest ranking males. Maybe they could be caught by the radical feminists as a symbol, right?
Social relationships among hyenas may have to do with maintaining the hierarchy, or to find food (hunting or scavenging), or reproduce, or control of the territory against other clans, and are based on communication systems that manifest with multiple sensory modalities, both body language and vocalizations. Of these, a wide range of sounds (about 12 different) have been registered, the best known of which are a howl and a kind of laughing where the nickname comes from. Body language is also quite complex, with different attitudes and positions of the ears, tail, etc., sometimes similar to wolves.
Like primates, spotted hyenas recognize individual conspecifics, are conscious that some clan-mates may be more reliable than others, recognize foreign family groups and rank relationships among clan-mates, and adaptively use this knowledge during social decision making.
Creamy secretion of anal scent glands, and olfactory communication
The title of this blog post refers to a particular form of communication, but very common among these hyenas: a chemical signal, olfactively detectable. It is an odorous marking, with a smelly white creamy secretion, called paste, produced by a pair of anal sebaceous glands. This secretion is composed of lipid-rich sebum and desquamated epithelial cells. The paste is deposited on grass stalks, and produces a powerful soapy odour, which even humans can detect. They do it on several occasions, as when lions are present, or the males do it near the dens, and most often in their territory limits. Often, after the pasting, they scratch the ground with their front legs, which adds even more flavours that come from the secretions of their interdigital glands. Clans mark their territories by either pasting or pawing in special latrines located on clan range boundaries.
In addition, this odorous secretion is also part of usual greetings among members of the clan. So, two of the individuals are placed in parallel and in opposite directions from one another, lifting one leg back and smelling each other anogenital areas .
Spotted hyenas greeting one another. Photo: Tony Camacho, Science Photo Library
The scent of paste secretion
The major volatile constituents of paste are fatty acids, esters, hydrocarbons, alcohols and aldehydes. Collectively, they give paste a pungent, sour mulch odour that persists, detectable by the human nose, for more than a month after paste is deposited on grass stalks.
It has been shown that odour of spotted hyena paste varies based on the individual identity, sex and group membership of the scent donor. Hyenas’ group-specific odours, in particular, are due to underlying variation in the structure of short-chain fatty acid (mainly acetic, propionic and butyric acids) and ester profiles of paste.
These odorants are well-documented products of bacterial fermentation. These scent glands are warm, moist, organic-rich and largely anaerobic, and thus appear highly conducive to the proliferation of fermentative symbiotic bacteria.
Symbiotic bacterial communities that produce social odour of hyenas
The bacteria use protein and lipid of glands as substrates, producing odoriferous metabolites, which are used by their mammal guests as chemical signals. The bacterial communities differ according to the hyena individuals and especially to the clans, according to symbiotic microbial communities are slightly different among clans, they are group-specific. Bacterial communities arise from the contact between the hyenas of the same clan, as they share the same space and common areas where they deposit the paste secretion. Spotted hyenas frequently scent mark the same grass stalks as their clan-mates (i.e. overmarking), and they often do so in rapid succession to one another. Therefore, overmarking appears to be a viable pathway for the transmission of bacterial communities among members of hyena clans. Although average genetic relatedness within hyena clans is low, it is higher within than among clans.
This mechanism to explain the social scent specific for group has also been proposed for some other mammals such as bats (Eptesicus fuscus, Myotis bechsteinii) and badger (Meles meles), but precisely in the spotted hyena it has been well demonstrated recently in an article published by scientists from Michigan (USA) .
These authors have worked with anal scent secretions of female hyenas from Masai Mara reserve in Kenya. They have shown by electron microscopy the presence of bacteria in the paste.
Bacilli- and cocci-shaped bacteria surrounded by the paste secretion, with lipid droplets (asterisk) .
Bacterial DNA was extracted from samples of paste secretion and 16S rRNA genes were amplified and sequenced. Comparing the obtained sequences with data from GenBank ® (public database of genetic sequences, http://www.ncbi.nlm.nih.gov), different bacteria were identified. The genera found were some of the groups of gram-positive Actinobacteria (Corynebacterium and Propionibacterium) and Firmicutes (Anaerococcus and others), and some of the gram-negative group of bacteroides. While the types found were more or less the same in the different clans of hyenas, the proportions of bacterial types were significantly different according to the clan.
Propionibacterium, coloured electron microscopy image: Dennis Kunkel Microscopy, Inc./Visuals Unlimited, Inc. Other species of this bacterial genus producing propionic acid are involved in the production of Emmental cheese types.
So, using the latest molecular techniques, culture independent techniques and sequencing, this work  shows that symbiotic bacteria may be helpful to their animal guests, by increasing diversity of odoriferous signals available, with variability among hyenas’ clans.
Importance of symbiosis
This is a quite peculiar symbiosis of bacteria with mammals. But, as you know, most mammals, including us the humans, live with millions of bacteria inside, many of which are beneficial, like most that inhabit the digestive tract or other body parts, which constitute the so-called “microbiome”. The probiotics we eat with some fermented dairy products contribute to maintaining populations of these symbiotic bacteria.
More and more data on the importance of symbiosis in multiple aspects of living beings is being known, as well as symbiosis is a key factor in evolution. Just remember that the most likely hypothesis for the origin of the first eukaryotic cells (about 2000 million years), is that it was due to a combination of the two types of prokaryotes, bacteria and archaea. Some millions of years later, the two well known endosymbiosis took place in the eukaryotic cell: bacteria carrying aerobic respiration that gave rise to mitochondria, and photosynthetic oxygenogenic cyanobacteria that were the origin of chloroplasts in algae and plants.
Other important evolutionary symbiosis were the establishment of mycorrhizae between fungi and plants, which led to the colonization of land by these, or nitrogen-fixing bacteria (Rhizobium) with legume plants, or a group of organisms, lichens, which are symbiosis of fungi with some algae or cyanobacteria, and live in many very different and hostile environments. And many other cases of symbiosis between distinct species getting benefits because live together.
So, symbiosis is a good lesson from biological evolution: by cooperation, benefits for both participants are always obtained.
 Mills, G., H. Hofer (1998). Hyaenas: status survey and conservation action plan. IUCN/SSC Hyena Specialist Group.
 Theis, K.R., T.M. Schmidt, K.E. Holekamp (2012) Evidence for a bacterial mechanism for group-specific social odors among hyenas. Nature Scientific Reports 2, 615