Category Archives: Bacteria
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 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
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.
As I mentioned in this blog a year and a half before (August 30th, 2012 “Is there life on Mars?”), the Curiosity rover began to walk by Mars, specifically inside the Gale crater, carrying its sophisticated instruments to analyze rocks, soil and atmosphere, that is, a well equipped geochemical laboratory analyzing the surface of another planet for the first time. You can see a video of 2 minutes of the 1st year of Curiosity here. As you know, one of the objectives of this analysis is to find evidence of whether there were favorable conditions for life on Mars, albeit in very bygone eras.
The Curiosity rover is walking inside Gale Crater, east of Syrtis Major, the Mars most prominence visible through a telescope. Image NASA/ESA/Hubble.
Mars is a planet with some similar features on Earth, such as the length of day, slightly more than 24 hours, but the greater distance from the Sun and very tenuous atmosphere make the average temperature to be -63 ° C, with daily oscillations from +20°C to -140°C. Having no oceans, the land surface of Mars is equivalent to the Earth, because Mars is smaller.
Comparison of Earth and Mars. The diameter of Mars is 3400 km, approx half of the Earth’s, but excluding oceans, the solid surface is similar on both planets. Image taken from T.E. Harrison, New Mexico State University.
Despite the difficulties to demonstrate the current existence of liquid water on Mars, from some years ago evidences have been found that there was water in the Martian surface, and it is being confirmed that during the first thousand million years there was plenty of water on Mars (Remind that both Mars and Earth are about 4.5 billion years).
Appearance of a region of Mars indicating the flow of water in the distant past. Image taken from T.E. Harrison, New Mexico State University.
Water history in Mars, in billions of years. Image from T.E. Harrison, New Mexico State University.
Curiosity shows an “habitable” lake 3800 million years ago
Thus, Curiosity rover has been taking samples in various ways (see in Figure below the 5 cm holes taking samples) and analyzing them. Studying the data, scientists have been able to write a series of articles that have been published in Science and other prestigious journals.
Two 5 cm holes made by Curiosity rover sampling in Yellowknife Bay of Gale Crater on Mars (NASA/JPL-Caltech/MSSS)
Among other articles, by last May was already published one (Williams et al 2013) with observations that the material extracted from the Yellowknife Bay of Gale Crater shows textures typical of fluvial sedimentary conglomerates, with rounded pebbles that indicate fluvial abrasion, and by their characteristics a water flow of nearly 1 meter per second has been deduced, with a depth of near 1 meter. Therefore, when this area was formed the climatic conditions, with abundant rivers, would have been very different from the current hyperarid and very cold environment.
A few weeks ago, on December 9th, Science Online has released a special edition dedicated to the latest findings of Curiosity, with 6 articles asserting that at Gale Crater there would have been a lake theoretically habitable for some organisms.
In one of the articles, maybe the most relevant one, Grotzinger et al (2013) describe the sedimentary rocks found by the rover and demonstrate that it corresponded to an aqueous environment with neutral pH, low salinity and different redox states of Fe and S. The presence of C, H, O, S, N and P (i.e., all biogenic elements) is also shown, and the authors estimate that this favorable environment for life could have lasted some few hundred thousand years, and therefore it demonstrates the biological feasibility of this fluvio-lacustrine environment of post-Noachian Mars, that is, about 3800 million years ago.
Therefore, these scientists deduced that this lake had fresh water, a water we could drink, and that around this lake various fluvial and lacustrine environments were present. This environment should be habitable for some organisms, and very similar to some current earth environments. The commented chemical compounds would be suitable for the survival of bacteria chemolithotrophic bacteria, id est, those which use inorganic compounds (such as Fe or S) as an energy source and use CO2 as a carbon source. On Earth we have many kind of these microbes, such as iron bacteria (Thiobacillus) or sulfur bacteria (including some sulfur thermophilic archaea) and the nitrifying ones. Although most of these chemolithotrophic bacteria on Earth are aerobic because they use atmospheric oxygen as electron acceptor, this metabolism is also known in some anaerobes, such as those that do the anoxic ammonium oxidation (process “Anammox”), for example Brocadia anammoxidans, or the same nitrifying bacteria in anaerobic conditions.
Therefore, with this habitable lake for some time, and with other similar environments, we can not reject the possibility that living beings had been sometimes in Mars.
View from Yellowknife Bay in Gale Crater, looking W-NW. This area of sedimentary deposits was the bottom of a freshwater lake. Photo: NASA/JPL-Caltech.
Estimated size and shape of the lake that was in the Gale crater. There must have been other similar. The arrows indicate the direction of the alluvial fan that flowed into the crater from its wall. Photo: NASA/JPL-Caltech/MSSS.
Another aspect studied in this extra issue of Science is the possible presence of methane in the Martian atmosphere (Webster et al 2013). Just like on Earth, methane is a potential sign of biological activity in the past. Previous observations made from Earth or from the Mars orbit speculated on the presence of some 10 ppb of methane, and its subsurface biological origin was also discussed. Nevertheless, in the measurements made in situ by laser spectrometry of Curiosity rover using a specific spectral methane pattern, it has not been detected since the values found are lower than 1 ppb. This reduces the probability of recent methanogenic microbial activity on Mars and it is limiting the possible contribution of recent geological and extraplanetary sources.
Finally, in another article (Hassler et al 2013) about measures of cosmic rays on the surface of Mars that Curiosity has made over a year, models of radiation are elaborated, for the time calculation of possible subsurface microbial survival, and also for the preservation of organic compounds of biological origin finding them billions of years later. Unfortunately, the conclusion is that the powerful effect of cosmic rays would cause very difficult any of these possibilities. Unlike the Earth, the magnetic field of Mars is very weak and the atmosphere too, things that help bombardment of cosmic rays and the solar wind on the Martian surface. However, there is evidence that in the past Mars had a magnetic field more effective, so hope is not lost …..
Achenbach J. 2013. NASA Curiosity rover discovers evidence of freshwater Mars lake. The Washington Post, Health & Science online Dec 9.
Anderson PS. 2013. Curiosity rover confirms ancient martian lake. Spaceflight Insider online Dec 11.
Grotzinger JP et 72 al. 2013. A Habitable Fluvio-Lacustrine Environment at Yellowknife Bay, Gale Crater, Mars. Science DOI: 10.1126/science.1242777 online Dec 9, 2013.
Harrison, T.E., New Mexico State University: http://astronomy.nmsu.edu/tharriso/ast105/Mars.html
Hassler DM et 23 al. 2013. Mars’ Surface Radiation Environment Measured with the Mars Science Laboratory’s Curiosity Rover. Science DOI: 10.1126/science.1244797 online Dec 9, 2013.
Kerr, RA. 2013. New Results Send Mars Rover on a Quest for Ancient Life. Science Now News, online Dec 9, 2013.
Science Special Collection Curiosity 2013. Curiosity at Yellowknife Bay, Gale Crater. Online: http://www.sciencemag.org/site/extra/curiosity/
Webster CR et al 2013. Low upper limit to methane abundance on Mars. Science 18 October 2013, Vol. 342 no. 6156 pp. 355-357
Williams RME et 38 al. 2013. Martian Fluvial Conglomerates at Gale Crater. Science 31 May 2013, Vol. 340 no. 6136 pp. 1068-1072
All we that studied “Bios” probably remember two known aspects of the symbiotic relationships of plant roots with microorganisms:
1) The bacterial Rhizobium nodules on the roots of legumes (Figure 1). These bacteria, with the nitrogenase complex, are among the few organisms capable of fixing atmospheric N2 transforming it into organic nitrogen, which is used by the plant, and symbiotically, the plant provides organic compounds to the bacteria. Thanks to these bacteria, plants such as legumes do not require nitrogen fertilizers.
Figure 1. Rhizobium nodules
2) The mycorrhizae, that is, the symbiotic relationships between fungi and plant roots. The most commonly known are the mushrooms always associated with some trees (Figure 2), such as the Lactarius sanguifluus associated with pines. In fact, mycorrhizae are present in most plants. Through this symbiosis, the fungi receive organic nutrients of the plant, and this can capture more easily water and mineral nutrients (especially P, Zn and Cu) by means of the fungus. In addition, mycorrhizae increase the resistance of plants to diseases coming from the soil and facilitate them inhabiting badlands.
Figure 2. Mycorrhizae of mushrooms with trees. Image from Shannon Wright
But these are only the best known of the symbiotic relationships between microorganisms and plant roots. Indeed, as the soil is full of microorganisms, many of these, including bacteria, fungi, algae, protozoa or viruses, are beneficial, symbiotic or otherwise, for the plants. And what is biotechnologically more interesting, more potential applications of these microorganisms to benefit crop plants are being found, which can be a good alternative to the use of fertilizers and pesticides.
Different microorganisms can have direct positive effects on plant nutrition as nitrogen fixation, mineralization of organic compounds, and solubilisation of elements not available to the plant (such as phosphates, K, Fe), but also indirectly positive effects, such as the production of hormones and growth factors, or protection against pathogens (García 2013).
Thus, there is a growing interest in the biological control of plant pathogens. It has been proven that some of these pathogens are inhibited by antibiotics produced by microorganisms in the rhizosphere (Raaijmakers et al 2002). Bacteria are being used (bacterization) for some years in soil or with seeds or other plant parts, with the aim of improving the growth and health of the plant.
Some of the best known and used bacteria in this sense have been Bacillus and Paenibacillus. Several species of these genera of aerobic spore bacteria are abundant in agricultural soils and can promote plant health in different ways, suppressing pathogens with antibiotic metabolites, stimulating plant defence, facilitating nutrient uptake by the plant, or promoting symbiosis with Rhizobium or with mycorrhizae (McSpadder 2004).
The genus Paenibacillus was reclassified from Bacillus in 1993, and includes P. polymyxa, a species N2 – fixing, which is used in agriculture and horticulture. This and other Paenibacillus species give complex and regular colonial forms in agar, even surprising (Figure 3), which vary according to environmental conditions. For this, a self-organizing and cooperative behaviour between individual bacterial cells is needed, using a system of chemical communication. This bacterial social behaviour would be an evolutive precursor of multicellular organisms.
Figure 3. Colonies of Paenibacillus dendritiformis, 6 cm diameter each, branched (left) and chiral (right) morphotypes. From Wikipedia Creative Commons.
The colonization of plant roots by these bacteria has been demonstrated, and also that they do it by forming biofilms (Figure 4). The inoculation of these bacteria to the roots promotes the growth, as shown in peppers (Figure 5). This appears to be due to the nitrogen fixing bacteria, which increases the formation of plant proteins and chlorophyll, thus increasing photosynthesis and physiological activities. And on the other hand, it has been shown that these bacteria produce siderophores, which facilitate Fe uptake by the plant (Lamsal et al 2012).
Figure 4. Colonization of Paenibacillus polymyxa and biofilm formation on roots of Arabidopsis thaliana. Adapted from Timmusk et al 2005.
Figure 5. Promoting growth effect of peppers (Capsicum annuum) by inoculation with Bacillus subtilis (AB17) and Paenibacillus polymyxa (AB15), respect the non-inoculated control. From Lamsal et al. 2012.
Moreover, bacteria such as Paenibacillus can be effective against plant pathogens. For example, it has been shown that a strain of P. lentimorbus (B-30488r) reduces the incidence of disease done by the fungus Alternaria solani in tomato. It has been tested (Figure 6) that after inoculating with Paenibacillus a plant infected with Alternaria, resistance to the fungus was induced in the plant. The bacteria degraded the cell walls of the fungus and also inhibited it by competition of nutrients. In addition, it was found that Paenibacillus has no negative effect on the microbial population in the rhizosphere of tomato (Khan et al 2012). These treatments are a good alternative to the use of fungicides, avoiding the environmental and health problems of these compounds.
Figure 6. Schema of the influence of Paenibacillus lentimorbus B-30488r in the interactions of tomato plant with Alternaria solani, a fungus pathogen (Khan et al 2012).
Finally, these Paenibacillus can also be useful to avoid the transmission of human pathogens such as Salmonella through the crop plants. Indeed, on the east coast of the USA a few years ago were detected outbreaks of Salmonella on tomatoes due to contamination of water. When they analyzed the microbiome present in the roots of tomatoes and these were compared with those of other places where there were no Salmonella contamination occurred, it was found that these tomatoes of the East Coast had no Paenibacillus, which were present in tomatoes of other places. With this, they decided to inoculate tomatoes with several Paenibacillus and found that Salmonella disappeared. Among the inoculated strains, one was selected as more effective, P. alvei TS -15 , for which a patent was obtained as a biocontrol agent of foodborne human pathogens (Brown et al. 2012) .
Thus, knowledge of the soil microbiota and the many forms of relationships between microorganisms and plants lead to find new strategies for using “good” microbes to prevent food safety problems of transmission of pathogens, while at the same time it can be a good ecological alternative to the massive use of pesticides.
Brown EW, Zheng J, Enurach A, The Government of USA (2012) Paenibacillus alvei strain TS-15 and its use in controlling pathogenic organisms. Patent WO2012166392, PCT/US2012/038584
Conniff R (2013) Super dirt. Scientific American 309, sept, 76-79.
Conniff R (2013) Tierra prodigiosa. Investigación y Ciencia 446, nov, 68-71.
García, Sady (2013) Los microorganismos del suelo y su rol en la nutrición vegetal. Simposium Perú “Manejo nutricional de cultivos de exportación”. Slideshare.net
Khan N, Mishra A, Nautiyal CS (2012) Paenibacillus lentimorbus B-30488r controls early blight disease in tomato by inducing host resistance associated gene expression and inhibiting Alternaria solani. Biological Control 62, 65-74
Lamsal K, Kim SW, Kim YS, Lee YS (2012) Application of rhizobacteria for plant growth promotion effect and biocontrol of anthracnose caused by Colletotrichum acutatum on pepper. Mycobiology 40, 244-251.
McSpadden Gardener BB (2004) Ecology of Bacillus and Paenibacillus spp. in agricultural systems. Phytopathology 94, 1252-1258
Raaijmakers JM, Vlami M, De Souza JT (2002) Antibiotic production by bacterial biocontrol agents. Antonie van Leeuwenhoek 81, 537-547
Timmusk S, Grantcharova N, Wagner EGH (2005) Paenibacillus polymyxa invades plant roots and forms biofilms. Applied and Environmental Microbiology 71, 7292-7300
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.