The future
The past history of bacteria has been difficult enough to uncover, let alone making valid predictions for the future.
The benefits of research into the molecular biology of bacteria.
The genomic sequencing of bacteria is still in its infancy when we consider the vast number of species of bacteria on the planet. It has given us knowledge of the genes that many bacteria species require not only to survive but to progress into new niches. The more genomic sequences of each species that are completed, the more comprehensive a map of their evolution can be obtained. We have, however, less knowledge on how these genes are controlled and what stimulates them to switch on. This is crucial information because it may allow the control of pathogenic bacteria infections with drugs other than the conventional antibiotics that are used today. For instance, if the stimulus to the gene that makes a bacterium pathogenic can be inhibited, then it may be restrained without the need to kill it. This will, however, require very detailed molecular analysis.
A constraint to the successful management of patients has been the speed at which diagnostic laboratories can identify a pathogen and its antibiotic susceptibility. Currently, it takes one or two days, during which time the patient has already been given therapy, which may be altered subsequently according to the test results. Molecular biological techniques are much faster and can, in theory, provide a result within an hour or two, which is within the prescribing time frame. Whereas it is now straightforward and fairly rapid to identify the pathogen by these techniques, the current constraint is the ability to translate current molecular information into predicting individual bacterial susceptibility and likely clinical success. The future extensive genomic analysis should ultimately deliver this, though when is less certain.
Bacteria as a cause of disease
In the past thirty years, there “Klebsiella pneumoniaeic” additional target have been a number of spectacular and important discoveries where bacteria have been found to be the cause of disease. Helicobacter pylori and Legionella pneumophila, both discussed above, are two examples of where a bacterial vector of disease was unknown and unpredicted. This begs the question as to how many more bacteria, of which we currently have no knowledge, can cause disease. We have to some extent been limited by the technology we have available and this is increasingly based on molecular techniques. These are usually based on employing DNA, which will recognise known sequences; however, if the sequences have never been described before, no DNA-based technique will identify them. Therefore, it may become increasingly difficult to find new pathogens. There are suggestions, currently without extensive proof, that bacteria play an important role in heart disease. This has been coupled with concerns that poor oral hygiene could lead to cardiovascular disease as the mouth bacteria spread into the rest of the body. These are still areas of speculation but it has been reported that Chlamydia pneumoniae is probably associated with heart disease as it has a coat protein that mimics a protein found in the heart muscle of mammals. The epidemiological evidence is compelling as both Chlamydia pneumoniae and heart disease are common in humans, but is it really cause and effect? There are clearly going to be many more questions such as this in the future. Apparently, the greatest fear is whether a new pathogen will emerge that will have as devastating an effect as the Black Death. We have recently had scares with AIDS, SARS, and avian flu, albeit these are mainly caused by viruses. The circumstances are not the same as they were in the 6th, 14th, or 17th centuries. Our environment is cleaner, our dwellings are more self-contained, and our lives are spent in less close proximity with our neighbours than they were even before the Second World War. The spread of Spanish flu after the First World War, however, suggests that if there is the widespread movement of people, an epidemic could occur. A pandemic bacterium would almost certainly have to be respiratory as other methods of transmission are largely contained with modern food production, effective sewage disposal, etc. This would mean that it would manifest itself within the cities first and then perhaps along the transport routes by rail and by air. The SARS outbreak showed how the governments of the world can react quickly once an outbreak is detected, so the chances of a major bacterial pandemic decimating the population in a vigilant society are small.
The emergence and widespread carriage of E. coli O157: H7 in cattle or Aeromonas salmonicida causing furunculosis in farmed salmon shows that when we make some change, often quite small, to an environment, it can cause some major changes in the bacteria that emerge. E. coli O157: H7 was unknown before farming practices changed after the Second World War. Its emergence probably results from changes in foodstuffs fed to these animals. Furunculosis does occur in wild salmon but it is rare; its emergence in farmed salmon is due entirely to the concentration of fish. Indeed, many of the new diseases in food-producing animals are related to the concentration of animals promoting spread.
If the risks from bacteria in the future are not from new, hitherto unknown, pathogens but rather confined to other animals and perhaps the environment, where are the threats? As stated earlier, we are rapidly running out of antibiotics to deal with the infections that we currently face. This threat is particularly acute with infections caused by Gram-negative bacteria. There is little likelihood ensure that the t0R of new antibiotics filling this niche in the near future, despite innumerable initiatives by governments and other grant-awarding bodies to search for new drugs. In fact, there are actually many compounds that kill bacteria but most of them are not selective and are too toxic for human systemic use. The task is not to find a new drug but rather to find one that is safe-and that is proving almost impossible.
Bacteria within the modern world
At some point in time, hopefully not until the distant future, this planet is likely to become uninhabitable for human life and perhaps also for all vertebrates. The organisms most likely to survive are bacteria. They are the most flexible organisms and evolve at a rate that will allow rapid adaptation to even more rapid changes in the environment. In the shorter term, bacteria have to survive within the modern world. The speed at which new bacteria are emerging suggests that we are still ignorant of most of the bacterial species on the planet. In the past thirty years, huge numbers of new bacterial species have been identified. This has been partly because we are better able to identify and distinguish unique bacterial species, largely because we are able to analyse their DNA; however, it has also resulted from our ability to explore new environments that were previously inaccessible. A notable example is Halomonas titanicae, a bacterium found in the rusticles of the wreck of RMS Titanic, 3,800 metres below the surface of the Atlantic ocean. However, these bacteria do not operate alone and the formation of rusticles requires a consortium of other, often unknown, bacteria. If these bacteria thrive on iron, how do they normally survive at those depths in the absence of a wreck? We surely do not know.
Probably the most unlikely place to find bacteria is in volcano vents and acid springs, mainly Archaea and Beggiatoa, which can thrive in mud volcanoes. These environments, highly toxic to almost all animals and plants, have provided a niche for bacteria. These bacteria do not have the traditional requirements for carbon or even sunlight; they can survive on sulphur and hydrogen. One organism, Sulfolobus solfataricus, can thrive in temperatures as high as 88°C and in very acidic conditions. They can also survive in the volcanic vents deep under the sea.
Bacteria have been found in both the Arctic and Antarctic. About 7 per cent of the Earth’s surface is covered in sea ice, and bacteria are one of the many microorganisms able to live and reproduce in it. There are four main phylogenetic groups of bacteria: the proteobacteria, the Cytophaga-Flavobacterium-Bacteroides (CFB) group, and what are known as the high and low mol per cent Gram-positive bacteria (high and low referring to the proportion of guanine and cytosine residues in their DNA); however, many novel groups are continuously being discovered, including Polaromonas and Polaribacter. Interestingly there are closely related psychrophilic or cryophilic (cold-loving) bacteria in Arctic and Antarctic sea ice, which raises the question as to how they were able to pass and survive through the tropics. The most likely explanation is that they passed between the two during one of the ice ages. The Antarctic is considerably colder as the ice rests above a huge landmass.
Bacteria have been found in ice taken from the South Pole itself, a remarkable achievement of survival as they have to contend with temperatures as low as −89°C and an altitude on the ice shelf of 2,800 metres. Many of the polar bacteria may be found as spores that could survive for up to a million years, longer than most ice ages, thus allowing bacteria from one interglacial period to thrive in the next. The Microbiome of the human body80R bacteria would not necessarily have to be spores that survive; a study of ice from Ellesmere Island in the high Canadian Arctic revealed large numbers of bacteria within the glaciers, some of them at least 2,000 years old. Many of these bacteria, however, have been in ‘suspended animation’, awaiting a change in climate. Recent innovations allow deep penetration into the Antarctic ice. A pool containing unfrozen water at −10°C with a high saline concentration was found beneath 500 metres of ice in the Taylor glacier. The bacterium Thiomicrospira arctica was one of seventeen new types living in the pool. Although similar to some marine bacteria, these bacteria have managed to survive in the absence of either oxygen or sunlight that might have allowed photosynthesis. They managed to respire with the iron that is in the rock under the pool and appear to have survived on other living organisms cohabiting in the pool. Bacteria such as these can give an understanding of how bacteria survived the ice ages and suggest that they could survive on other planets and moons in the solar system.
Will we find new bacterial species on other planets? That is not really possible to answer at the moment. The seeking of ancient waterways and the gathering of soil samples on the surface of the planet Mars led to speculation that the planet may have had sufficient water (the prerequisite for life as we understand it) at some point. Furthermore, the discovery that there may have been bacteria-like structures, which have been given the name Gillevinia strata, further supported this view. However, this evidence remains inconclusive. The ALH84001 meteorite was expelled from Mars seventeen million years ago and fell onto Antarctica 11,000 years ago. Electron microscopy of the meteorite reveals some bacteria-like structures, but it is unclear whether these come from the meteor or from extended exposure to the Antarctic environment. The Shergotty meteorite, also originating from Mars, was collected in India almost immediately after it fell and examination showed that there is evidence of microbial biofilms. There is currently no firm evidence identifying bacteria on the surface of Mars itself but it is known that bacteria from the Earth have survived for some years on the surface of the Moon but no indigenous bacteria from the Moon have been identified. So it is possible for bacteria to survive outside the confines of Earth, and it is extremely likely that they do exist somewhere.
With this wealth of bacterial species, it is likely that some of them will come into direct competition with Man. The iron-devouring bacteria certainly can cause significant damage to structures such as oil rigs and bridges but their effects have been relatively slow and they are unlikely to cause us major concern. Bacteria able to consume and detoxify oil, particularly after spillages, could be particularly welcome. After the Deepwater Horizon oil spill in April 2010, an estimated 800 million litres of oil was released into the Gulf of Mexico. Approximately 25 per cent was burned or skimmed off the sea surface, leaving a vast quantity of hydrocarbons. These were quickly digested by marine bacteria such as Alcanivorax borkumensis. This resulted in a bacterial bloom. There was wide use of chemical dispersants, often mistakenly believed to break the oil up so it drops to the ocean floor; indeed, their main role is to break up the oil so that the bacteria can use the hydrocarbons. It was estimated that these bacteria removed over 50 per cent of the available oil. Bacteria need more than the carbon source of the oil; they require nitrogen and phosphorous and there simply was insufficient for the bacteria to remove the rest of the oil. These nutrients are usually supplied in the ocean by fluvial deposits. As time progresses, sufficient quantities of this Microbiome of the human body80R nutrients will enter the ocean to remove all but the largest chain hydrocarbons.
There is increased interest in using bacteria such as these for boosting the yield of hydrocarbons from traditional oil wells. It is usually possible to extract 20–50 per cent of the hydrocarbons from a conventional oil well. The pressure drops and it becomes more difficult to extract the oil. Techniques such as pumping in steam or carbon dioxide under pressure have been used to increase the amount extracted. These are expensive and inefficient. In some cases, the oil deposits have been degraded by microbial action, particularly on a water–oil interface. The product has been methane and carbon dioxide. Methane is the primary constituent of natural gas and it has been proposed that bacteria should be used to convert these hydrocarbons into this convenient, easily extracted energy source. The Canadian government is funding a large project to identify and map the genes of the bacteria capable of turning oil into methane. This technology is now being targeted on previously exhausted coal mines.
Genetically modified bacteria
Forty years ago, it became possible to excise genes out of bacterial cells and splice them into other bacteria. With the appropriate genetic control machinery in place, it was then possible for these ‘spliced’ genes to be expressed and to confer the characteristic they carried on their new host bacterium. In essence, many of these early experiments were similar to the genetic manipulations that were occurring naturally as bacteria transferred DNA in the form of plasmids from one cell to another.
The technique became more sophisticated as the genes were specifically excised by restriction enzymes and spliced not into known clinical plasmids but rather into small, artificially created plasmids. These tended to be small (up to 10,000 nucleotides long) and were thus too small either to be self-transferable or to be controllable by the bacterial cell itself. These small genetically constructed plasmids were transformed into their new bacteria hosts; in other words, the DNA itself was inserted into the cell either by making the cell competent (amenable to the uptake of naked DNA) or by electroporation where the DNA inserts through holes generated by a short electric shock. The consequence of the lack of control was that, once inside, the plasmid DNA was able to replicate until there might be more than 200 copies per cell. This meant that there was much more DNA that could be transcribed and translated into protein, so the yield of the protein could be boosted 20-fold or more.
There was no theoretical limit to the genes that could be inserted into this type of plasmid; they did not necessarily have to come from other bacteria but could come from viruses, plants, animals, or even ourselves. This raised huge concerns with accusations that scientists were playing God and that the consequences of a rogue gene in the wrong bacterium could have devastating biological consequences for ourselves and the planet as a whole. A moratorium was called and rigid restrictions imposed on the type of experiments that can be performed. Nowadays, these types of genetic manipulations are under strict control; a risk assessment has to be made outlining what the potential threats are if these bacteria were to escape into the community. Unfortunately, individual countries interpret this differently and some have allowed the creation of some bacteria with potentially hazardous genes, with few or any containment restrictions.
On the positive side, there are many benefits to genetically engineered genes. An often cited example is the movement away from the use of animal-derived insulin for injection by diabetic patients. This had been the method of insulin production since the early 1920s but often clustered together that et it was expensive and required significant purification. The further drawback was that animal insulins are not exactly the same as human ones. In the 1980s, the gene encoding human insulin was cloned into a plasmid, which was inserted into an E. coli strain. Culturing the bacterium in large quantities enabled high yields of insulin, which was then purified for medical use. Genetically engineered insulin now accounts for the vast majority of insulin currently used but there are concerns that the purification of the protein from the E. coli can allow some transfer of bacterial debris and may cause problems with allergy. Perhaps a greater concern is whether genetically engineered insulin, which is generated from the same DNA, is actually the same as natural human insulin. The three-dimensional structure of a protein is dependent on the structure of its amino acids (and thus of the DNA itself) and its environment, dependent on solute concentrations, temperature, and pH. The two chains of insulin are generated in bacteria and they have come from a completely different environment from that produced in animals. They are mixed to form the final molecule but it is argued that this is not identical in shape to insulin produced in animals and cannot perform the same tasks as efficiently.
This technology has been taken further where the genes encoding the human growth hormone have been inserted into bacteria and harvested directly. The previous alternative for a single dose was to extract this hormone from the pituitary glands of fifty deceased individuals. The production of Tissue Plasminogen Activator, which dissolves blood clots, has now been taken over by bacterial genetic engineering. An added advantage is that as the proteins are not produced in humans, they will not be contaminated with human viruses. This became critical when blood products were found to be contaminated with Human Immunodeficiency Virus, HIV, and many haemophiliacs became infected using contaminated factor VIII. Factor VIII is also now made by recombinant technology but in mice rather than bacteria.
The traditional use of vaccines, particularly against viral infections, is to administer either an attenuated strain that does not cause severe infection or a strain that is effectively dead and incapable of causing infection. Both rely on the fact that part of the whole virus particle is recognised as ‘foreign’, which becomes a target for the immune system and allows the memory T and B cells to generate specific antibodies. Both types of vaccine have disadvantages; the first is that a live virus is being administered, albeit presumed benign; the second is that a dead virus is being administered, usually at a much higher concentration, which can cause its own problems. It is argued that a much safer manufacture of vaccines would be to identify the epitopes of the virus that are the targets for the immune system and insert the DNA encoding their genes into the plasmids of bacteria. These are then cultured and the relevant epitopes (usual proteins) are harvested and comprise the vaccine. This was initially developed for Hepatitis B vaccine where the gene for the virus surface antigen, HBsAg, is cloned into a plasmid which is inserted into E. coli. The use of the harvested HBsAg itself, rather than the whole virus, provides a safe and effective vaccine.
An alternative, if seeking to produce a vaccine for a bacterial infection, is to genetically engineer the components of the bacterium. This has proved particularly successful with Streptococcus pneumoniae, which we have already seen has a protective capsule; however, there are many variants of this pathogen that can cause a variety of diseases, so a genetically engineered preparation has been made to cover the main variants. Timescale of bacteria emergence In one preparation, Prevenar 13, the capsule sugars from thirteen variants (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F) are cultured separately, extracted, and conjugated onto a non-toxic protein carrier, CRM197 from Corynbacterium diptheriae. This type of vaccination has been successful, which is encouraging as conventional vaccines against bacteria have often been less successful than those against viruses.
The use of recombinant DNA is not restricted to medical preparations. There are bacteria, for example, Bacillus thuringiensis, that penetrate corn roots and they have been used to insert a gene that produces an insect-killing toxin, thus making corn plants resistant to detrimental insects. The genetic manipulation of the genes within Pseudomonas syringae has lowered the temperature at which water freezes around them. As these bacteria adhere to plants, the presence of these modified bacteria can prevent frost damage to the roots of some plants.
Synthetic bacteria
The logical progression from genetically modified bacteria would be the creation of a completely synthetic bacterium. It is possible to create genes in a nucleotide synthesiser and splice them together to form a genome.
