A quorum of flavour – Part 1
Gut flora bacteria. Photo: 123rf.com
As a lover of cheeses, it is quite relaxing for me to research how cheeses are created – and in particular, the complex mix of bacterial and fungal cultures needed to create the idiosyncratic characteristics and flavours of various cheeses.
One of my favourite cheeses, St Nectaire Fermier, is produced by an astonishingly complex and active bacterial community made up of debaryomyces hansenii, geotrichum candidum, brevibacterium linens, torulopsis sphaerica, kluyveromyces lactis, candida sake, cladosporium, aspergillus, etc. Some of these same bacteria are also active in the production of blue cheeses as well, with the characteristic blue furrows developed by either penicillium roqueforti or penicillium glaucum.
What was immediately curious is how these various microbial cultures manage to co-exist so peacefully in the cheeses. It would be more plausible to assume that one of the more aggressive organisms would attempt to wipe out the other species and dominate the whole curdy landscape – and very likely introducing a single sour taste to all cheeses.
But it seems that these various bacterial and fungal colonies are perfectly happy to co-exist in cheeses – acting rather like a perfect multi-racial human society where each race contributes subtly yet significantly to the cultural flavour and well-being of the country.
Obviously, this utopian situation very rarely happens, even with seemingly intelligent human beings, so how come simple bacteria and fungi are so much better at co-existence?
The answer is rather fascinating and also explains why our bodies tolerate the many billions of bacteria in the microbial flora in our intestines.
Some bacteria behaviour: The bacteria in the culture on the left are sensitive to the antibiotics contained in the white paper discs. The bacteria on the right are resistant to most of the antibiotics. (The bacteria are Escherichia coli.) Photo: Wikimedia Commons/Dr Graham Beards
Some evidence of bacterial co-operation was already observed in the mid-1960s, notably by a Hungarian-born scientist called Alexander Tomasz. By 1994, scientists had determined that bacteria can indeed detect variations in their environment caused by other bacteria – what’s more, the bacterium can understand the implications of such changes and apply specific survival strategies based on the environmental alterations.
Quorum Sensing
A key piece of research penned in 1994 by the American scientists, Fuqua, Winans and Greenberg, sketched out the concept of Quorum Sensing (QS) by bacteria. The original research was based on marine luminescent bacteria but the principles of QS (or inter-bacterial communication) has since been validated and found to apply to many other mixed colonies of bacterium, including those in cheeses.
At the simplest level, QS is managed by the production and interplay of groups of signalling molecules known as autoinducers – the individual molecules are known as inducers and they are detected by special receptors in the bacterial cells.
A single cell can emit one or more different kinds of inducers. The interaction between inducers and receptors can be quite simple or very complex, depending on various factors, not least being the proximity of other colonies of different bacterium in the same environment.
There are three general types of inducers – the ones produced by Gram-positive bacteria are based on peptides (or chains of amino acids) while Gram-negative bacteria use derivatives of fatty acids.
If you’re curious, Gram-positive bacteria have thicker cell walls which can retain the purple dye used in the Gram stain test (hence the stain effect is positive) – so by inference, Gram-negative bacteria are not stained by the Gram stain test and that’s because their thinner cell walls cannot hold the stain.
Gram positive spherical-shaped bacteria. Photo: Filepic
The final type of inducers is rather rarer and can be produced and utilised by both Gram-positive and Gram-negative bacteria – these inducers are unusual because they are based on boron, an element seldom associated with biomolecules.
Regardless of the types of inducers, the effect at the cell level is like a non-linear equation; that is, the effect can become suddenly chaotic.
However, it is important to note that autoinducers work not only on external bacterium but also within the same colony of bacteria. Within the same colony, the bacteria may choose to wait and expand until the colony reaches a certain size before breaking out of their enclave – this is how many bacterial infections begin.
The bacteria are always aware of its colony size by the amount of autoinducers around it and won’t break out until it is confident of overwhelming the host’s body defences.
The interaction between different species of bacteria goes along somewhat similar lines. Let’s review a situation where the numbers of autoinducers from an external bacterium are slowly growing in an environment colonised by another single host species of bacteria.
While the numbers of external autoinducers are small, nothing much really happens – the inducer molecules are detected, counted by the host receptors and things continue quietly between both colonies of bacteria. And it stays that way until the quantity of inducers detected reaches a threshold (also known as the quorum).
Once the quorum is reached, the host bacteria react rather more frenetically and in several ways – one way is by activating a gene which produces more receptors.
Another way is to activate a separate gene which promotes the production of its own autoinducers – almost like a signal to alert its presence to other bacterium.
There are then several options available to the host bacteria – they can attempt to destroy the external invading bacteria, they can choose to tolerate each other as peacefully as possible, or they can form a partnership with the external bacterium.
Gut flora
Within our own bodies, there are examples of all three kinds of behaviour. Foreign virulent bacteria (also known as pathogens), once detected in the blood or organs, are set upon by the white blood cells in a violent attempt to kill the invaders.
However, in the gut, the externally-introduced microflora work together magnificently with the body – in fact, much of the body’s immune system emanates from, or is enhanced significantly, by the bacterial colonies there.
Probiotics, the natural friendly bacteria are an integral part of the digestive system. Photo: Filepic
And within the gut bacteria, there are also countless species which don’t do much for the body but are tolerated either because they are food for the good bacteria or they just don’t have much impact on anything and it is too difficult to rid them from amongst the rest of the gut microflora.
So although there are many kinds of autoinducers swirling around in the gut, it does seem that the various species of gut bacteria generally tend to get along fine with each other.
Back to the cheese
So from the above, you can probably surmise, quite correctly, that the multiple colonies of bacteria and fungi in cheeses also tolerate each other pretty well. The main difference is that they don’t have any noble aims to benefit a higher organism (as with human gut microflora) – most of the bacteria in cheeses just co-exist together in blocks of curdled milk, producing their individual microbial end products, and the final results are the idiosyncratic flavours and textures of cheeses.
As an aside, if you have been drinking alcohol, the ethanol can significantly impact the microflora in the gut, mainly by killing them. Hence it is always a good idea to ingest some probiotic products the next day to replace the lost bacterium. The alternative, of course, is to avoid drinking alcohol but that would be as ridiculous as the idea of me converting to a religion.
Probiotics need prebiotics
It should be noted that it is also quite pointless to ingest probiotics if you don’t provide them with the food they need to survive. Therefore it is a good idea to snack on some prebiotic foods before having the probiotics – the bacteria in the probiotics need the complex carbohydrates (eg. oligosaccharides and inulin) in prebiotic foods to feed on.
So even if you don’t drink alcohol, the microflora in the gut can still die off over time if they are not supplied with prebiotic carbohydrates. Some useful prebiotics are found in tasty ingredients such as mushrooms, leeks, green bananas, okra, onions, garlic, beans, etc.
However, the story actually doesn’t end so simply. What’s quite curious is that certain bacteria appear to understand the principles of QS and hence are able to work around QS – a classic example is a potentially dangerous bacterial species known as staphylococcus aureus. These bacteria appear to know how to avoid producing autoinducers that can be detected by other organisms until it is too late – one reason might be because the genus staphylococcus is often tolerated as part of the normal bacterial flora on human skin and does not get dangerous until it enters the body.
Bacterial behaviour is like… corrupt politicians
It is somewhat like the blatant abuse of trust in political corruption – a corrupt politician, after winning the trust of the people, probably starts on the road to corruption with tiny, imperceptible frauds before expanding to working with other dishonest people and companies to steal vast amounts of money from the public they are supposed to serve. Therefore, there are significant similarities in the behaviours of certain human criminals and pathogenic bacteria.
As an aside, in the case of staphylococcus aureus, there is a deadly strain called methicillin-resistant staphylococcus aureus (MRSA) which is extremely resistant to antibiotics – once contracted, the prognosis for the patient is usually rather poor and the irony is that this strain is quite prevalent in the areas where antibiotics are commonly used, such as hospitals. This is the strain that eventually killed my father.
Hospital-associated Methicillin resistant Staphylococcus aureus (MRSA) bacteria. Photo: Filepic
Bacteria cities and towns
Hiding isn’t the only strategy for invasive bacteria – there are other strategies and a study of these techniques may lead to new innovative treatments to combat infections. It may have other important applications, such as the delay or prevention of food decay – or even improve dental hygiene.
Like humans, many bacteria appear to appreciate that the overall chances for survival can be significantly higher if they organised themselves as communities. Freely floating bacterial cells are known as planktonic cells – and can be individually targeted by the body’s defence systems.
However, as communities, the various species of bacteria can share and benefit from the attributes of each of the species in the community while working under a common protective shield.
A community of such groups of different bacteria is known as a biofilm – and a classic easily visible example is the plaque on teeth.
A biofilm can be considered like a small town or city full of pathogens interacting and communicating with each other via autoinducers. As such, it has been suggested that the autoinducers do more than just signalling a presence – they also appear to be the communication medium for organising the communal work needed for the survival of the biofilm.
The alternative theory is that bacteria sensing the denser presence of another more virulent pathogen would tend to join and support the stronger colony rather than risk annihilation.
More and more other bacterial species would also join the more powerful pathogens, thereby creating a multi-species colony – and eventually the presence of the other bacterium controls the growth and behavioural characteristics of each individual species.
Curiously, this behaviour is also predicted in Hamilton’s Rule for sociobiological altruism which is paraphrased a little as follows: rb > c, where c is the cost of being cooperative, b is the fitness or survival benefit conferred to the bacteria and r is the relatedness or compatibility of the cooperating bacteria to the overall colony.
So it seems that bacteria can also solve some basic mathematical equations. Whatever is next?
http://www.star2.com/living/viewpoints/2016/03/13/curious-cook-a-quorum-of-flavour-part-1/This post is on Healthwise
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A quorum of flavour – Part 2
MARCH 27, 2016
BY CHRIS CHAN
Electron microscope view of bacteria attached to tooth enamel, prior to forming plaque. Filepic
Regardless of how various types of pathogens decide to cooperate in the creation of a biofilm, the stages for the formation of a biofilm always follow five well-defined steps.
Step 1 – Reversible attachment to a surface
Bacteria first adhere very tenuously to tissue surfaces, using weak, reversible bonds known as van der Waals forces.
These forces are quite interesting in that they operate purely between molecules, even if the molecules are neutral and have no inherent charge.
The weakest van der Waals force is known as the London dispersion force, which arises from the very temporary and spontaneous charge that occurs as electrons move around the atoms in the molecules.
A stronger van der Waals force is the dipole-dipole force, which relates to polar molecules with an unequal sharing of electrons.
An example is hydrochloric acid (HCl) where the hydrogen is positively charged as the chlorine atom has pinched an extra electron from the hydrogen atom (hence making the chlorine atom negatively charged).
In a solution of such molecules, the van der Waals forces will make the hydrogen poles of HCl molecules line up with the chlorine poles of other molecules.
The strongest van der Waals force is hydrogen bonding – this is exactly the same as the dipole-dipole force except that it specially relates to any bond between a hydrogen atom and either oxygen, fluorine or nitrogen.
Why it is special is because hydrogen is quite keen to lose electrons when bonded with oxygen, fluorine or nitrogen which in turn are very keen to attract electrons – hence these compounds have both extremely positive and extremely negative polar molecules.
This is one reason why these compounds (eg. water) tend to make good solvents – they can grab and hold molecules of other soluble compounds in between the solvent molecules.
It is important to note that the van der Waals forces relate only to the attraction between complete molecules only – they are not chemical reactions in any way. They are also the most important natural forces in the world – without van der Waals forces, life could not have evolved on Earth.
Step 2 – Irreversible attachment to a surface
Eventually, the pathogens that have managed to remain attached start to construct exopolymeric structures – these are effectively cages made of polysaccharides which are either synthesized by the bacteria themselves or scavenged from the body.
While the pathogens are free-floating or planktonic, they are very prone to being detected and destroyed by the body’s white blood cells (phagocytes). Under the exopolymeric shelter or nascent biofilm, the pathogens are sheltered and less prone to being attacked.
Step 3 – Formation of micro-colonies
One or more species of bacteria now move into the developing biofilm and begin the process of colonisation, extending the exopolymeric structures further to accommodate the growing pathogenic population. The phagocytes are still in attendance but are more and more thwarted by the protection provided by the biofilm.
Step 4 – Formation of a mature biofilm
The biofilm develops into a well-ordered three-dimensional sessile structure, with clusters of bacterial cells arranged between channels that allow the transport of water, nutrients and the disposal of waste material. The word sessile simply means “established” or “not able to move about”.
The increasingly desperate phagocytes now attempt to destroy the biofilm by releasing copious amounts of phagocytic enzymes.
Step 5 – Detachment and dispersal of cells from a mature biofilm
It has been suggested that once a new quorum in the biofilm is reached, the pathogenic cells will burst out from the biofilm as planktonic cells to launch virulent attacks on parts of the body. These pathogens may also be migrating with the intention of forming new biofilms.
The dispersal of pathogens may or may not be assisted by the actions of the phagocytic enzymes themselves, which can weaken the tissue structures around the biofilms, thereby provoking the leakage of pathogens.
What happens to teeth
It might be interesting to investigate how biofilms actually work in real life – and a good example would be the biofilms that cause tooth decay, especially as some 92% of the US population suffers from some form of dental decay. And dental decay is simply the end result of the presence of residue carbohydrates, particularly sugars, in the mouth and teeth. The groundwork for the biofilm that eventually becomes plaque on human teeth is probably laid by the bacterial genera neisseria and streptococci.
Both genera are interesting in other ways – besides the formation of dental biofilms, the neisseria family contain pathogens which are responsible for meningitis and gonorrhoea. The genus neisseria is also curious as several strains exhibit a survival technique known as antigenic variation, which is the ability to alter its surface proteins in order to evade the body’s defences – it is like a bacterial chameleon.
Formation of Biofilms, by D. Davis – From: Looking for Chinks in the Armor of Bacterial Biofilms, Monroe D PLoS Biology Vol. 5, No. 11, e307 doi:10.1371/journal.pbio.0050307
There are some 25 strains of streptococci present in the human oral cavity and different strains help with the various stages of biofilm formation as well as actual tooth decay – in fact, streptococci mutans even has special receptors which significantly improves the ability of this bacteria to adhere to teeth.
After the initial groundwork for the biofilms, the early exopolymeric biofilm structures are colonised by lactic acid-producing strains of lactobacilli and other streptococci. It is at this stage that the formation of visible plaque begins – bacteria related to lactobacillus reuteri secrete an enzyme called glucansucrase which converts sucrose into carbohydrates, which are then used as scaffolding to attach more and more of the bacteria to the tooth enamel.
At the same time, bacteria similar to streptococci mutans (eg. streptococci sobrinus) also digest sucrose using an enzyme called dextransucrase, producing a sticky polysaccharide made of dextran which is also attached to the teeth – a polysaccharide is effectively a complex carbohydrate chain consisting of simple sugars.
The resulting layer of carbohydrates created by the lactobacilli and streptococci bacteria is what we see as plaque. The position of the plaque places the pathogens on the surface of the teeth where the lactic acid produced by the bacteria breaks down teeth enamel (which mainly consists of hydroxy-apatite, also known as crystalline calcium phosphate) in a process called demineralisation – and demineralisation is the actual cause of tooth decay.
If you’re curious, lactic acid is produced by lactobacilli from its homofermentative metabolism and streptococci derives its acid from the anaerobic fermentation of carbohydrates (in particular, sugars) – note that lactic acid has a pH of 4 but the pH can be lowered further by the consumption of acidic drinks or foods.
Gum disease may arise due to the constant acidity around the teeth weakening the gum tissue or perhaps also due to the persistent presence of phagocytic enzymes on the gums as the body’s defences attempt to attack the plaque bacteria.
Don’t pick nits
You might have heard comments like “sugars don’t cause tooth decay, it is acid that causes teeth to rot” – but that is as ridiculous as the Americans saying, “guns don’t kill people, people kill people”.
It is just a form of nit-picking, and you can create other fatuous lines like “guns don’t kill people, bullets kill people” or “guns don’t kill people, the trauma of lead projectiles entering the body at a terminal velocity of 1,150 metres per second kill people”.
The reality is that the bacteria that cause tooth decay are always present in our mouths and all they need to grow, multiply and cause dental decay is the presence of carbohydrates, particularly in the form of sugar.
The decay of foods, biofilms and quorum sensing
Everyone knows that food decays because of the presence and actions of bacteria – and these bacteria normally rely on biofilms to breed and propagate. One main reason is because foods seldom have moving channels of blood or fluids and therefore planktonic dispersal is highly improbable. Instead, bacteria contaminating food tend to inch along by enlarging established biofilms – this is easily visible if you make a time-lapse series of photos of a rotting fruit over a period of days.
Research suggests that reducing sugars added to sugar-sweetened drinks by 40% during a five-year period could prevent 1.5 million cases of people becoming overweight and obese. Photo: Courtesy Fotolia/TNS
There has been a lot of study into how to stop or delay the formation of biofilms in food – if a method can be found to prevent the rotting of food, the savings to the food industry would be incalculable. It will also significantly reduce the incidence of food poisoning, if there is only some way to prevent pathogens building biofilms from which to spread.
Hamilton’s Rule may have some bearing on the direction of the research into the prevention of biofilms. You will recall that the (paraphrased) rule is: rb > c, where c is the cost of being cooperative, b is the fitness benefit conferred to the bacteria and r is the relatedness or compatibility of the cooperating bacteria to the overall colony.
The factor r, is where quorum sensing comes in. If there is some way to mess with the autoinducers and impair the attraction of different pathogens to working with each other, then the cost of being cooperative for each species of bacteria would be too high and therefore the pathogens would never come together.
The idea to write something about quorum sensing started while enjoying the miracle of a lovely piece of St Agur cheese on a ficelle, as I sipped some nice claret. So now you know how some things work at the microbial level.
Before ending, I have one last important piece of cheese advice: If you leave cheese too long anywhere, it will develop discolorations, white patches and/or mould.
If this happens, it means that one or more biofilms have formed on the cheese and hence the cheese is now no longer a happy community of various cheese-flavouring bacteria.
As most biofilms are pathogenic (ie. full of toxin-producing microbial organisms), the best (and smartest) thing to do would be to throw away the cheese immediately.
Sometimes I do scrape off the discoloured bits and eat the rest but that is simply because I can be a little reckless – there is no need to follow my bad example. In fact, like a lousy politician, I advise against following my own bad behaviour.
Just as an addendum, some very recent research has found that certain soil bacteria (specifically bacillus subtilis) can also communicate using electrical signals, not unlike the type of signalling which happens in our brains – this was something thought only possible for multi-cellular organisms.
Whether some of the bacteria in our bodies or in biofilms can also do this is not known at present – but it is humbling how simple organisms like bacteria can still continue to surprise us, even now.
