18th August 2019
Translated from the original article in Catalan
A few months ago -April 2019- my friend Jordi Diloli, Professor and Archaeologist, shared a very surprising article (Aouizerat et al 2019) with me. It was echoed on the internet (Borschel-Dan 2019), and I will comment here.
“Resurrected” yeasts from 3,000 years ago
The group of researchers led by Ronen Hazan of the Hebrew University of Jerusalem took samples of 21 clay containers from various sites in present-day Israel from 2500 to 5000 years ago, from the Persian, Philistine and Egyptian (this is the oldest) periods. Archaeologists believed that these vessels contained fermented beverages such as beer or mead (Figure 1). The authors submerged the containers in a rich YPD medium, specific for growing yeasts and other fungi, and incubated them at room temperature for 7 days. Then, samples of this medium were spread on agar plates with the specific medium, and the resulting colonies were isolated for subsequent analyses (Aouizerat et al 2019).
Figure 1. Clay vessels from where the yeasts were isolated (Image of Judah Ari Gross, Times of Israel).
The isolates that were found were 6 strains of different yeast species, and one of which was Saccharomyces cerevisiae, specifically from a Philistine site dated 3000 years ago. Obviously, it is very surprising that living yeasts of such ancient remains have been isolated. For this reason, the authors of the work carried out a series of experiments that could confirm this unique fact and that the isolates were not a product of contamination.
Firstly Aouizerat et al (2019) showed that it is possible to isolate yeasts from clay vessels that have contained beer or wine after a certain time. They did so with containers with unfiltered beer buried for 3 weeks, and also with another vessel that had repeatedly contained wine but not used last 2 years. With these samples they developed the isolation methodology and in both cases they were able to isolate yeasts. No isolates were obtained from a control sample with filtered beer, therefore without yeasts.
To demonstrate that the isolates were originals of the old vessels because these had contained the fermented beverage, authors applied the same protocol with samples of other ceramics that were surely not for this purpose, and also with sediments near the containers. The result was clearly negative for these samples: only 2 isolated yeasts from 110 samples, while the mentioned 6 yeast strains were isolated from the 21 initial samples. That is, yeasts would be significantly more abundant in containers of alcoholic fermented beverages than in other related archaeological vessels or sediments around them.
Another argument that supports the hypothesis of this work was the identification of these 6 yeasts. Total DNA was obtained and processed to sequence the genomes and compare them with the databases. Two of them, from the Egyptian period, were identified as Saccharomyces delphensis, a species that has been isolated from African dried figs and is not at all common on soil. Therefore, this suggests the use of figs in the alcoholic beverages of these containers. Another isolate was identified as Rhodotorula, common pollutant yeast in African beers. Another was identified as Debaryomyces, a frequent yeast in traditional African sorghum beers. As said before, another isolate was identified as Saccharomyces cerevisiae, the yeast most used to make wine, beer or bread (Figure 2). In spite of this, the genetic sequence of this S. cerevisiae was clearly different from the strains most commonly used today, as commercial or laboratory strains, and therefore the possibility of contamination is excluded. And finally, the other isolate was identified as Hypopichia burtonii, previously isolated yeast from Ethiopian mead.
These genetic data, together with the phenotypic characterization -fermentative kinetics and other biochemical characteristics carried out with the isolates by Aouizerat et al (2019)- suggest that these yeasts actually come from an environment related to alcoholic beverages. These authors even elaborated beer with these isolates and some of them, especially the Saccharomyces, gave a very good analytical and sensory result.
Figure 2. Saccharomyces cerevisiae at the scanning electronic microscope (MD Murtey & P Ramasamy)
Aouizerat et al (2019) conclude that the isolates are descendants of yeasts that were originally used 3000 years ago, in large quantities and in repeated fermentations. This would have facilitated their survival in pore microenvironments of the ceramic matrix of these containers, and the microcolonies would have continued to grow minimally for millennia thanks to the humidity and residual nutrients. The authors make the analogy with some handmade beers where it is usual that the containers waste serve as starter for new productions.
Finally, the authors of this work speculate that it is possible to isolate microorganisms from archaeological remains, not only yeasts, and that in the case of bacteria it could even be easier, given the resistance characteristics of some of them, such as the sporulated ones.
Is there no previous similar work to that of Aouizerat et al (2019) ?
As we have seen, this is certainly a very surprising finding. Scientifically, the work is quite accurate and has been “approved” by the international community: the article is published in an open-access journal with prestige (mBio, high impact factor: 6.7), of the American Society for Microbiology, where all the articles are reviewed by a minimum of two experts, besides the editors. The results presented by the article seem very well worked, and the conclusions are well reasoned.
However, in my opinion it is still almost incredible, and it is strange that nothing like this has been found before. Maybe if someone else had previously tried to isolate such old microorganisms without getting them, perhaps it would not have been published ? Maybe nobody has previously tried to do something similar ? A “malicious” explanation might be that archaeologists have their own interests and microbiologists or molecular biologists have others, and that for this type of work the collaboration of both is needed. Well, it seems not being so, since there are a lot of studies on microorganisms from ancient remains, but they have been almost always focused on the detection and analysis of ancient DNA. These studies demonstrated the presence of certain microorganisms although they did not proceed to isolate them.
DNA gives evidence of microorganisms in ancient remains
In relation to yeasts, the oldest evidence is that ribosomal DNA of Saccharomyces cerevisiae has been obtained from residues found in Egyptian wine jars 5000 years old (Cavalieri et al 2003). It must be remembered that the oldest archaeological evidence of large-scale wine production has 7400 years, in north of the Zagros Mountains, in present-day Iran (McGovern et al 1986). As it is known, S. cerevisiae is also the bread and beer yeast, derived from cereals, but since neither S. cerevisiae nor its spores are aerial, surely the use of this yeast in fermented grape juice, as well as dates, figs or honey, historically preceded its use for brewing and bread (Cavalieri et al 2003). It is probable that the wine yeasts naturally occurring in damaged grapes (Mortimer & Polsinelli 1999) were used to ferment other cereal products such as cereals, and after centuries of selection for humans, they evolved into specific strains to ferment food and beverages from cereals.
The genomes of pathogenic microorganisms have also been studied in archaeological remains by means of new massive DNA sequencing techniques, in order to know to epidemic diseases of historical importance, such as black plague, tuberculosis, cholera or leprosy (Andam et al 2016). Logically, in these cases the archaeological remains are human ones, such as bones, teeth, coprolites or mummified tissues. In this way, for example, the phylogeny and evolution of Yersinia pestis strains causing the black plague have been recognized by remains of the Bronze Age (5000 years ago) and until the well-known epidemics of the 6th and 14th centuries (Bos et al 2011). Another well-known case is the Helicobacter pylori genome identified in the intestine of the Ötzi mummy, the iceman in the eastern Alps, 5300 years old (Maixner et al 2016).
DNA has also been isolated from specific bacteria of the human gut, such as Bifidobacterium and Bacteroides, to demonstrate the human presence in archaeological sediments 5000-12000 years old, in north east of Poland (Madeja et al 2009).
It should be remembered that DNA is degraded over time, and in fact it is more unstable than other cellular components. This macromolecule spontaneously suffers damage by oxidation, hydrolysis, and fragmentation in pieces that may be less than 100 bp. Most fossils or other biological remains of more than 100,000 years old no longer contain PCR-amplifiable DNA (Hofreiter et al 2001), although it seems that if the samples are extracted from frozen sediments, with constant temperatures below zero, DNA could be recovered from up to 400,000 years or a little longer (Willerslev et al 2003). In addition the tissues are colonized over time by fungi and bacteria that greatly reduce the relative amount of endogenous molecules and can contribute to giving false positives. The risk of contamination is very high and often this is not taken in account. Generally the DNA of the host that is analysed can be less than 1% of the total DNA found. All these factors complicate the DNA extraction, the construction of sequence libraries, the alignment of DNAs and the analysis of genomes (Andam et al 2016).
Surprisingly, there are a few published works where it is found old DNA of plants, animals and various microorganisms, some million years (My) old, even hundreds of My. The most remarkable are those obtained from amber samples of 20-40 My, and those obtained from a halite 250 My old. This would be comparable to the Jurassic Park fiction where almost non-degraded DNA from the dinosaurs of 100 My old “was recovered”.
Hebsgaard et al (2005) thoroughly reviewed all these more spectacular cases, with the conclusion that these works suffered from inadequate experimental approaches and inadequate authentication of the results. Therefore, there are great doubts as to whether DNA sequences and in some cases viable bacteria could survive such large geological times.
In addition, it is worrying that these works with so old DNA have not been replicated independently in order to confirm their authenticity, and that they did not show a relationship between the age of the sample and the persistence of DNA depending on the different types of bacteria (Willerslev et al 2004). In contrast, Willerslev et al studied the persistence of DNA in permafrost and they found a clear relationship of DNA degradation with time (Figure 3). As seen, DNA amount is very small beyond 100,000 years and it is hardly found beyond 1 My.
Figure 3. Persistence of not degraded bacterial DNA over time (kyr, thousands of years) maintained in permafrost, measured by fluorescence (Willerslev et al 2004).
When analysing the bacterial phyla of these DNA, Willerslev et al (2004) observed (Figure 4) that the most persistent are those of Arthrobacter, the main representative of Actinobacteria (high G+C gram-positive), followed by sporulated (Bacillaceae and Clostridiaceae), and finally the Gram-negative Proteobacteria.
Figure 4. Proportions of the main bacterial phyla (Actinobacteria in brown, sporulated in orange and Proteobacteria in blue) based on DNA obtained from permafrost samples, along time (kyr, thousands of years) (Willerslev et al 2004).
This increased persistence of non-sporulated Actinobacteria is surprising because sporulated bacteria have always been considered the most resistant of all types of cells. Although endospores have special adaptations such as proteins binding DNA to reduce the rate of genetic modifications, they do not have active metabolism or repair and their DNA will degrade over time. The mechanism of greater resistance of Actinobacteria is unknown, but there may be some activity and repair of DNA at temperatures below zero, and/or adaptations related to the dormant cells state (Willerslev et al 2004).
Anyway, the limit for PCR-amplifying the DNA would be between 400,000 years and 1.5 My for samples kept below zero, but this is much more unlikely in non-frozen materials, such as the amber of halite samples of million years, and much less likely to find viable cells from these samples so old (Willerslev et al 2004).
The same commented works where DNA of some millions of years (My) was found, are the most surprising cases of having “resurrected” microorganisms, basically bacteria: viable cells of the sporulated Bacillus from amber samples of 30 My (Cano & Borucki 1995), Staphylococcus also from amber of about 30 My (Lambert et al 1998), and the most spectacular case of Bacillus from an halite of 250 My (Vreeland et al 2000 ). This sporulated bacterium would have been in a hyper-saline environment of the last Permian and trapped in a salt crystal, surviving until now. In the case of Staphylococcus isolated from amber, in spite of not being sporulated, they are bacteria very resistant to extreme conditions, and which have been isolated also from ancient permafrost and very dry environments (Lambert et al 1998).
In spite of this, the revision of these cases by Hebsgaard et al (2005) concludes that none of them fulfilled the relative rate of molecular distance test, which is the probable rate of mutations calculated in comparison to related lineages. Therefore, these isolations are arguable and not reproduced. In addition, in the case of the 250 My Bacillus, it has been argued that the inclusion of bacteria in the halite could be the consequence of a subsequent recrystallization (Lowenstein et al 2011).
Another review on microorganism preservation records (Kennedy et al 1994) comments published cases up to 600 My, indicating that it is curious that there are several cases with more than 1 My, and also cases with less than 10,000 years ago, but there are very few cases of intermediate periods. These authors also point out the doubts raised by works with surviving bacteria so old, which would surely be artefacts or contaminations.
On the other hand, the most credible works are those of Abyzov et al (2006) and Soina et al (2004), which demonstrated the presence of several living microorganisms, both prokaryotes and eukaryotes (especially yeasts, but also some microalgae), in Antarctic ice samples that have some thousands of years. These authors combined classical microbiological methods, such as enrichment and isolation of colonies, together with epifluorescence microscopy, electronic microscopy, and molecular techniques. The bacteria found were Gram-positive (Micrococcus) and gram-negative (Arthrobacter), which are not sporulated, but they have cist-shaped dormant cells, which can survive while maintaining viability at temperatures below 0ºC for some thousands of years.
When geologically ancient DNA findings are published as well as viable cultures of ancient samples, the independent reproduction of the results by another laboratory is fundamental, to exclude any contamination from the same laboratory. In the case of having recovered living cells, it is necessary to demonstrate the reproducibility of the isolation, sequencing the genomes of the cultures obtained in independent laboratories from the same sample, and checking that in both cases the genomes coincide (Hebsgaard et al 2005).
From the remains of the Roman fort of Vindolanda, in the north of England, viable endospores of Thermoactinomyces, member of Bacillales (Unsworth et al 1977) have been recovered. They are about 1900 years old and the remains were a mixture of clay with straw and other vegetable materials. The authors propose to use these sporulated bacteria as indicators in archaeological studies.
Besides sporulated bacteria, there are several groups of non-sporulated ones for which anabiosis resistance abilities have been demonstrated. In particular, they have been isolated from permafrost and the tundra soil of Siberia of about 1 My (Suzina et al., 2006), in the limit of what we mentioned earlier (Willerslev et al 2004), which is quite difficult to believe. In order to study experimentally the formation of these anabiosis forms, Suzina et al incubated several gram-positive and gram-negative bacteria, and some archaea, in poor media with limiting nitrogen, and after a few months they obtained their dormant cells. They had cist structures, with capsule and a thickened cell wall, intramembranous particles and a condensed nucleoid (Figure 5). They also observed that these cysts did not have metabolic activity and supported stress factors such as lack of nutrients or heating.
Studying the permafrost isolates, they confirmed that there are cist structures very similar to those obtained in the laboratory, with multi-layer wall structures of up to 0.4 μm. In fact, these authors believe that most of the bacteria present in the permafrost and the tundra are in the form of a cyst (Suzina et al 2006).
Figure 5. Sections of a vegetative cell (a) of Micrococcus luteus and of a cyst cell (b) of the same bacterium, obtained after 9 months of culture in a medium limiting in nitrogen. C, microcapsule; CW, cell wall; OL1, 2, 3, outer layers of the cell wall; IL, inner layer of the wall; CM, cytoplasmic membrane; N, nucleoid. The bar measures 0.3 μm (Suzina et al., 2006).
Other “resurrected” yeasts and fungi
Besides the surprising mentioned article by Aouizerat et al. (2019), there are other few published cases of yeasts and other “resurrected” fungi such as the following.
Chicha is a beer-like beverage from corn, yellowish and slightly effervescent, elaborated and consumed by Andean populations for some thousands of years, whose traditional process has the peculiarity of using amylase of saliva for convert the starch into fermentable sugars. Fermentation traditionally took place in clay containers called “pondos”. From the remains of the chicha pondos from the Hipia culture in Quito (2100-2800 years old), various yeast were isolated, especially Candida, Pichia and Cryptococcus (Gomes et al 2009). Interestingly, some of these yeasts have been confirmed molecularly as Candida theae, similar to those isolated from contaminated Asian tea (Chang et al 2012). It is worth mentioning the absence of Saccharomyces in these ancient chicha, although today it is the main yeast, coming probably from beer and wine fermentation that led the Spaniards (Gomes et al 2009).
From Greenland ice samples of about 100,000 years (Ma et al 1999), several microorganisms were revived, such as bacteria (Micrococcus, Rhodotorula, Sarcina) and yeasts (Candida, Cryptococcus) and other fungi (Penicillium, Aspergillus). The authors also isolated the DNAs and demonstrated the phylogenetic relationship of the isolates. Once again, we see how ice provides a stable environment that facilitates the conservation of microorganisms and their DNA.
Raghukumar et al (2004) have recovered living Aspergillus (sporulated Ascomycota fungus) and other fungi from sediment samples of the deep-sea, about 5900 m deep in the Chagos trench, south of the Maldives, in the Indian Ocean. Based on the depth in the sediment and the present Radiolaria, authors estimated that they correspond to a minimum of 180,000 years, and up to 430,000 years in some samples. From the isolates identified as A. sydowii they obtained spores that germinated and grew in hydrostatic pressure equivalent to the depth of 5000 m, and at a temperature of 5ºC. With microscopy of epifluorescence and bright field, the fungal hypha and their relation to the particles of the sediment are clearly observed (Figure 6). It seems that this Aspergillus found in the deep-sea is the oldest fungus recovered alive so far. The authors suggest that preservation would have been possible thanks to high hydrostatic pressure, along with low temperature.
Figure 6. Photomicrographies of deep-sea sediment (5900 m) of the Indian Ocean with hyphae of Aspergillus sydowii and sediment particles. (a) epifluorescence microscopy combined with that of bright field; (b) epifluorescence (Raghukumar et al 2004).
One of the most surprising works, and hard to believe, is that of Kochkina et al (2001), where a lot of fungi of all kinds and bacteria, especially actinobacteria, were isolated from samples of permafrost from Russia, Canada and Antarctica reaching 3 My old. The authors even suggested that there is no limit of years to recover viable microorganisms. This article has had very little echo, and it is not even mentioned by later articles as Raghukumar et al. (2004).
As we have seen, evidence of DNA from no-living yeasts in ancient remains related to winemaking dates back to around 5000 years in ancient Egypt (Cavalieri et al 2003). Regarding other microorganisms, taking into account the natural degradation of DNA over time, it seems that the oldest samples would be about 400,000 years at most, in particular actinobacteria in frozen sediments such as permafrost (Willerslev et al 2003 ). Publications of bacterial DNA recovered from several millions years (up to 600 My) have many scientific concerns about their credibility and reliability (Kennedy et al 1994).
With regard to living yeast as those of 3000 years apparently isolated by Aouizerat et al (2019), it seems that Candida and others were isolated from containers to elaborate chicha about 2800 years old (Gomes et al 2009), although this reference is a review and the original work does not appear to have been published. Other authors (Abyzov et al 2006; Soina et al 2004) also find alive yeasts, without specifying which ones, in Antarctic ice samples of some thousands of years. More surprising are the isolated isolations of yeast and other fungi and bacteria from Greenland ice samples 100,000 years old (Ma et al 1999), as well as those of Aspergillus from the Indian Ocean seabed of about 180,000 years (Raghukumar et to 2004).
Regarding other “resurrected” microorganisms, some of the most reliable are the several Antarctic ice bacteria of some thousands of years (Abyzov et al 2006) and Thermoactinomyces spores of Roman remains 1900 years old (Unsworth et to 1977). Of the oldest, perhaps the anabiosis forms of bacteria conserved in permafrost a million years old (Suzina et al., 2006) would have certain likelihood. Curiously, these bacteria would be non-sporulated but they would have a cyst structure, with multi-layer walls and other intracellular modifications. The other findings of “resurrected” bacteria from more millions of years of amber or halite, just like their DNA and also because of this, are very hard to believe (Hebsgaard et al 2005).
Thinking in the cellular forms of resistance and anabiosis, as the bacterial endospores and the mentioned cysts, it must be remembered that yeasts, like many other fungi, have the ability to produce spores, in particular ascospores as they are Ascomycetes. Although these ascospores have a greater capacity for resistance than vegetative cells in dry conditions or other inhospitable environments and have a persistence in time, apparently there is no work (or I have not been found) related to the recovery of yeasts ascospores from ancient remains.
The work of Aouizerat et al (2019) makes no mention of the yeast spores, neither as a possible explanation of the yeast survival in these ancient remains. In fact, they propose that the microcolonies of yeasts on ceramics pores would have continued to grow minimally during these 3000 years thanks to the humidity and residual nutrients. Well, we do not know, and neither if the yeast ascospores have had any role.
Finally, we can believe the finding of Aouizerat et al (2019) is truth, but obviously further investigation in other similar archaeological samples must be done. This research should be done not only for yeasts, but also for bacteria of other fermented products. Besides considering the sporulated ones, other bacteria should be considered, that could survive thanks to the cell cysts or other forms of anabiosis.
Abyzov SS et al (2006) Super-long anabiosis of ancient microorganisms in ice and terrestrial models for development of methods to search for life on Mars, Europa and other planetary bodies. Adv Space Res 38, 1191-1197
Andam CP et al (2016) Microbial genomics of ancient plagues and outbreaks. Trends Microbiol 24, 978 –990
Aouizerat T et al (2019) Isolation and characterization of live yeast cells from ancient vessels as a tool on bio-archaeology. mBio 10, 2, 1-21
Borschel-Dan A (2019) Israeli scientists brew groundbreaking “ancient beer” from 5,000-year-old yeast. The Times of Israel, 22nd may 2019.
Bos KI et al (2011) A draft genome of Yersinia pestis from victims of the Black Death. Nature 478, 506–510
Cano, R.J. and Borucki, M.K. (1995) Revival and identification of bacterial spores in 25- to 40-million year-old Dominican amber. Science 268, 1060–1064
Cavalieri D et al (2003) Evidence for S. cerevisiae fermentation in ancient wine. J Mol Evol 57:S226-232
Chang CF et al (2012) Candida theae sp. nov., a new anamorphic beverage-associated member of the Lodderomyces clade. Int J Food Microbiol 153, 10-14.
Gomes FCO et al (2009) Traditional foods and beverages from South America: microbial communities and production strategies. Chapter 3 in Industrial Fermentation, ed. J Krause & O Fleischer, Nova Science Publishers.
Hofreiter M et al (2001) Ancient DNA. Nature Rev Genet 2, 353–359.
Kennedy MJ et al (1994) Preservation records of micro-organisms: evidence of the tenacity of life. Microbiology 140, 2513-2529.
Kochkina GA et al (2001) Survival of micromycetes and actinobacteria under conditions of long-term natural cryopreservation. Microbiology 70, 356-364
Lambert LH et al (1998) Staphylococcus succinus sp. nov., isolated from Dominican amber. Int J Syst Bacteriol 48, 511-518
Lowenstein TK et al (2011) Microbial communities in fluid inclusions and long-term survival in halite. GSA Today 21, 4-9
Ma L et al (1999) Revival and characterization of fungi from ancient polar ice. Mycologist 13, 70-73.
Madeja J et al (2009) Bacterial ancient DNA as an indicator of human presence in the past: its correlation with palynological and archaeological data. J Quaternary Sci 24, 317-321.
Maixner F et al. (2016) The 5300-year-old Helicobacter pylori genome of the Iceman. Science 351, 162–165
McGovern PE et al (1986) Neolithic resinated wine. Nature 381:480–481
Mortimer R & M Polsinelli (1999) On the origins of wine yeast. Res Microbiol 150, 199-204
Raghukumar C et al (2004) Buried in time: culturable fungi in a deep-sea sediment core from the Chagos Trench, Indian Ocean. Deep Sea Res Part I: Oceanog Res Papers 51, 1759-1768
Soina VS et al (2004) The structure of resting microbial populations in soil and subsoil permafrost. Astrobiology 4 (3), 345–358.
Suzina et al (2006) The structural bases of long-term anabiosis in non-spore-forming bacteria. Adv Space Res 38, 1209-1219.
Unsworth BA et al (1977) The Longevity of Thermoactinomycete Endospores in Natural Substrates. J Appl Microbiol 42, 45-52
Vreeland RH et al (2000) Isolation of a 250 milion-year-old halotolerant bacterium from a primary salt cristal. Nature 407, 897-900.
Willerslev E et al (2003) Diverse plant and animal DNA from Holocene and Pleistocene sedimentary records. Science 300, 791-795
25th December 2018
Translated from the original article in Catalan.
We humans are destroying the planet Earth. Besides climate change (there are still ignorant people who do not believe it), the depletion of natural resources and the massive extinction of animal and plant species, one of the most visual effects is the coverage of the planet with rubbish. Since 71% of the surface is marine, most of the non-degrading waste finishes in the sea. In the oceans there are already large expansions covered by floating debris, especially plastics, called “plastic islands” (Figure 1). In the North Pacific area, where different sea currents come together, the “island” reaches 1500 km of radius, with plastics up to 200 meters deep, and continues to grow. There is more information of it, and also about the environmental consequences, in the Wikipedia article Great Pacific garbage patch.
Figure 1. Small portion of the Great Pacific Garbage Patch (From oceanandreserveconservationalliance.com)
Although there are many types of plastics, one of the most used and most abundant in waste and “plastic islands” is polyethylene terephthalate, known as PET or PETE (Figure 2). It is a type of thermoplastic polymer, vulgarly plastic, which belongs to the so-called polyesters, and is obtained by synthesis from petroleum. It is harmless, very resistant and lightweight and has multiple applications (Figure 3). Counting only bottles of PET for refreshing beverages, 1 million of them per minute are sold in the world. It is a recyclable material (see Pet bottle recycling in Wikipedia) but very resistant to biodegradation. In nature it can last some hundreds of years.
Figure 2. PET, polyethylene terephthalate.
Figure 3. Several applications of PET (From http://www.technologystudent.com).
PET is “eaten” by Ideonella sakaiensis
I. sakaiensis (Figure 4) are bacteria with rod shape, gram-negative, non esporulate aerobic heterotrophic, mobile with a flagellum, and catalase (+) and oxidase (+) (Tanasupawat et al 2016). They grow at neutral pH and are mesophilic, with optimum at 30-37°C. They belong to the phylogenetic group of betaproteobacteria, which include, besides many others, the known Neisseria (gonorrhoea and meningitis) and the nitrifying Nitrosomonas.
Figure 4. Scanning electron microscope images (false colour) of Ideonella sakaiensis cells grown on PET film for 60 h (From Yoshida et al 2016).
The 201-F6 strain, the first of the new species I. sakaiensis, was isolated from a landfill and identified in 2016 by a Japanese group of the Kyoto Institute of Technology that looked for bacteria using plastic as carbon source, from samples of remains of PET bottles (Yoshida et al 2016). They saw that these bacteria adhere to a low-grade PET film and can degrade it, by means of two enzymes characterized by these authors: a PETase and a MHETase, which produce terephthalic acid and ethylene glycol acid (Figure 5), which are benign environmental substances and that the bacteria can be metabolized. A colony of I. sakaiensis completely degraded a low-grade PET bottle in 6 weeks. High-grade PET products need to be heated to weaken them before the bacteria can degrade them. This is the first bacterium found as a PET degrader, and uses it as the only carbon source and energy source. Since PET has existed only for 70 years, these bacteria should have evolved in this short period until being able to degrade PET in a few weeks, instead of hundreds of years in nature (Sampedro 2016).
Figure 5. Predicted metabolic pathway of PET degradation by I. sakaiensis: extracellular PETase hydrolyses PET giving monohydroxyethyl terephthalic (MHET) and terephthalic acid (TPA). MHETase hydrolyses MHET to TPA and ethylene glycol (EG). The TPA is incorporated through a specific transporter (TPATP) and is catabolized to cyclohexadiene and this to protocatechuic acid (PCA) by the DCDDH. Finally, the PCA ring is cut by a PCA 3.4 dioxygenase with oxygen, as known for degradation of phenolic compounds and other xenobiotics. The numbers in parentheses are the ORF of the corresponding genes (From Yoshida et al 2016).
Previously, only some tropical microfungi (Fusarium solani) were known to degrade PET, and they also excreted esterases. In this case, Fusarium would be used to modify the polyester fabric, to achieve more hydrophilic and easier to work (Nimchua et al 2008). It is important to remember the structural similarity of synthetic PET fabrics (Figure 3) to those of natural fibre such as cotton, since these contain cutin, which is a polyester, a waxy polymer from the external parts of the plants. Therefore, the enzymes of Fusarium or Ideonella must be relatively similar to those that were already in nature long before the plastics were invented.
Recent genetic improvement of the enzyme PETase of Ideonella sakaiensis
In order to better understand the function and specificity of the PETase, a group of American and British researchers have recently characterized the structure of this enzyme (Austin et al 2018), mainly by high resolution X-ray crystallography, comparing it with a homologous cutinase obtained from actinobacteria Thermobifida fusca. The main differences between the two have been a greater polarization in the surface of the PETase (pI 9.6) than in the cutinase (pI 6.3), and on the other hand (Figure 6), a greater width of the active-site cleft in the case of PETase of I. sakaiensis. The cleft widening would be related with an easy accommodation of aromatic polyesters such as PET.
Figure 6. Compared structures (left) of the PETase of I. sakaiensis (above) and the cutinase of actinobacterium Thermobifida fusca (below), obtained by high resolution X-ray crystallography (0.92 Å). The active-site cleft is marked with a red dotted circle. Details (right) of the active site with different cleft widths in the PETase of I. sakaiensis (above) and the cutinase of T. fusca (below) are shown. (From Austin et al 2018).
Hypothesizing that the structure of the active site of the PETase would have resulted from a similar cutinase in an environment with PET, Austin et al (2018) proceeded to make mutations in the PETase active-site to make it more similar to cutinase and obtained a double mutant S238F/W159H which theoretically would make the entry of the active site closer (Figure 6). But their surprise was capital when they saw that the mutant degraded the PET better (an improvement of 20%), with an erosion of the PET film (Figure 7 C) even greater than the original PETase (Figure 7B). The explanation was that mutant changes in amino acid residues favoured PET intake in the active site, despite making a closest cleft (Austin et al 2018).
Figure 7. Scanning electronic microscopy images of a piece of PET without microorganisms (A), after incubating 96 h with PETase of the I. sakaiensis 201-F6 (B), and with PETase of the double-mutant S238F/W159H (C) (From Austin et al 2018).
In addition, these authors have shown that this PETase degrades also other similar semi aromatic polyesters, such as polyethylene-2,5-furonicarboxylate (PEF), and therefore this enzyme can be considered an aromatic polyesterase, but it does not degrade aliphatic ones.
The conclusion of their work is that protein engineering is feasible in order to improve the performance of PETase and that we must continue to deepen in the knowledge of their relationships between structure and activity for the biodegradation of synthetic polyesters (Austin et al 2018).
Other plastic-eating microbes ?
The discovery of I. sakaiensis has been very important for the possibility of establishing a rapid recycling process for PET, but it is not the first organism that has been found as plastic consumer. By the way, we can see the formulas of the main plastics derived from petroleum in Figure 8.
Figure 8. Formulas of the most common petroleum plastics: polyethylene (PE), polyvinyl chloride (PVC), polypropylene (PP), polystyrene (PS), polyethylene terephthalate (PET or PETE) and polyurethane (PU) (From Shah et al 2008).
Reviewing the bibliography, we see that many cases of plastic degrading microorganisms have been described (Shah et al 2008), especially polyethylene, polyurethane and PVC: various Pseudomonas, Rhodococcus and Comamonas among bacteria, and some Penicillium, Fusarium and Aspergillus between fungi.
Among the polyurethane consumers, mushrooms are highlighted (Howard 2002), and especially the plants endophyte Pestalotiopsis microspora, which can use polyurethane as the only source of carbon (Russell et al 2011).
On the other hand, the ability of the mealworms, the larval forma of the darkling beetle Tenebrio molitor, to chew and degrade the polystyrene foam is well known (Yang et al 2015). Fed only with the PS, these larvae degrade it completely in relatively short periods. As expected, the degradation of the PS is carried out by the intestinal bacteria of the animal (Figure 9). It has been demonstrated because degradation stops when administering antibiotics to the larva (Yang et al 2015). One of the isolated bacteria that has been shown to degrade PS is Exiguobacterium, from Bacillales group, but it is not the only one. In fact, when performing studies of metagenomics from gut of larvae eating PS, a large variety of bacteria have been found, and these vary depending on the kind of plastic, since the degradation of polyethylene has also been seen. Some of the bacteria with DNA found as predominant would be the enterobacteria Citrobacter and Kosakonia. It seems that the intestinal microbiota of Tenebrio is modified and adapted to the different ingested plastics (Brandon et al 2018).
Figure 9. Biodegradation of polystyrene by the intestinal bacteria of Tenebrio, the mealworm (Yang et al 2015).
Finally, as we see the microbial biodegradation of non-biodegradable or recalcitrant plastics should not surprise us, since on the one hand, there are natural “plastics” such as polyhydroxybutyrate or polylactic acid that are easily degradable (Shah et in 2008), and on the other hand the adaptive capacity of the microorganisms to be able to break the most recalcitrant chemical bonds is very large. Microbes evolve rapidly, and acquire better strategies to break the plastics made by humans (Patel 2018). We have seen in this case the degradation of PET, which in less than 70 years some microbes have already found a way to take advantage of it.
The problem is that we are generating too much plastic waste in no time and the microorganisms have not had time yet to degrade them. It is clear that we will have to help our microbial partners, not generating more degrading polymers, and recycling and degrading them, by using these same degrading microbes, among other ways.
Austin HP et al (2018) Characterization and engineering of a plastic-degrading aromatic polyesterase. Proc Nat Acad Sci 115, 19, E4350-E4357
Brandon AM et al (2018) Biodegradation of Polyethylene and Plastic Mixtures in Mealworms (Larvae of Tenebrio molitor) and Effects on the Gut Microbiome. Environ Sci Technol 52, 6526-6533
Howard GT (2002) Biodegradation of polyurethane: a review. Int Biodeterior Biodegrad 42, 213-220
Russell JR et al (2011) Biodegradation of polyester polyurethane by endophytic fungi. Appl Environ Microbiol 77, 17, 6076-6084
Sampedro J (2016 marzo 10) Descubierta una bacteria capaz de comerse un plástico muy común. El País
Shah AA et al (2008) Biological degradation of plastics: a comprehensive review. Biotechnol Adv 26, 246-265
Tanasupawat et al (2016) Ideonella sakaiensissp. nov., isolated from a microbial consortium that degrades poly(ethylene terephtalate). Int J Syst Evol Microbiol 66, 2813-2818
Yang et al (2015) Biodegradation and mineralization of polystyrene by plastic-eating mealworms: Part 2. Role of gut microorganisms. Environ Sci Technol 49, 12087-12093
Yoshida et al (2016) A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 351,1196–1199
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
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