Humanity has forever been plagued by infections and has—for well over 2,000 years now—tried to fight them, beginning in the Iron Age with herbs and mantras, to vaccines and other complex drugs including antibiotics in the present day. From the rather simplistic belief that the human body was built of a few elemental Humours and that the disruption of the balance among them caused disease, to a deep understanding of the molecular processes that sustain life and bring it to states of disrepair, we have come a long way in these two millennia.

The formal development of the “germ theory of disease” in 17-19th century Europe, which posited that a certain class of diseases—referred to as “infectious diseases”—was caused by microscopic forms of life, was a cornerstone in the modern fight against these diseases.

That certain diseases were caused by invisible “animalcules” was first hypothesised in ancient Greece, medieval India and Arabia. These ideas resulted in the earliest forms of seasonal vaccination against diseases such as smallpox in medieval Asia. But post-Renaissance European involvement—including the participation of figures such as Louis Pasteur and Robert Koch—led to the development of vaccines against a variety of infections, culminating in the development of the first chemical therapeutic Arsphenamine or Salvarsan for syphilis in the 1910s; the craze for the effective but toxic sulpha drugs in the 1930s; and finally the discovery and industrial production of Penicillin G in the 1940s, heralding the birth of the Age of Antibiotics. The war against infections—bacteria in particular, against which antibiotics work—had been won.   

Five decades and many other antibiotics later, it’s dawned on us that bacteria have fought back. Many antibiotics are becoming ineffective as bacteria develop resistance to these magic bullets. Penicillin in its original form is nearly useless, except against a few exceptionally culpable bacteria. Deadly bacteria, including multidrug resistant Mycobacterium tuberculosis that causes difficult-to-treat but not necessarily untreatable tuberculosis, are becoming increasingly common. The recent identification of a multidrug resistant E. coli in the U.S. triggered an unusually large number of alarm bells and was interpreted by many as apocalypse. A frenetic search for new antibiotics is on. The British have called for “ancient-biotics”, describing a class of anti-infectives revived from the books of the ancients. Our own bhakts and vaidyas would be nodding sagely, “We told you so”. Life has come full circle. 

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hat are antibiotics? Antibiotics are chemicals that kill bacteria, or at least inhibit their growth, while being safe for human consumption. They do not affect other disease causing microorganisms such as viruses and fungi; hence the proliferation of public awareness posters on why antibiotics cannot cure you of a viral cold. They kill bacteria by interfering with cellular processes essential to their survival. For example, penicillin prevents bacteria from producing their cell wall, i.e. the cell component that gives a bacterium an identity by separating the inside of the cell from the outside. Without a cell wall there is no cell. Many variants of penicillin, including the much used and abused amoxicillin, are available in the market today. The antibiotic gentamicin inhibits protein synthesis. And rifamycin, used as a first line antibiotic against TB, inhibits the essential cell process called transcription by which information contained in the DNA is converted to RNA, a non-negotiable prerequisite for protein synthesis.

Antibiotics are largely safe for humans. Human cells are classified as “eukaryotes” and form an evolutionary branch distinct from bacterial cells, which fall under the category “prokaryotes”. Bacteria and humans, being evolutionarily distant, differ in many respects at the molecular level. For example, human cells have no cell wall, and are instead bounded by a “plasma membrane”. Bacteria too have fragile plasma membranes protected by the outer cell wall and in many cases by a second membrane. Thus an antibiotic that inhibits cell wall synthesis cannot hurt human cells. The protein synthesis machinery of humans and bacteria—despite sharing many commonalities—show several differences exploited in the design of antibiotics. Thus antibiotics are designed to target components essential to the life of a bacterium but not to humans.

With the success of penicillin came many antibiotics, whose use was not strictly regulated. Antibiotics did not seem to harm us so we did not hesitate to take amoxicillin for a cold without a doctor’s prescription, even though this was inappropriate. We even started using antibiotics to prevent, rather than cure infections; this practice took off in animal husbandry, which fostered antibiotic use to fatten livestock.

In a recent issue of their magazine Down to Earth, the Centre for Science and Environment has highlighted the widespread use of antibiotics in fish feed in India, a decades-long practice. To make matters worse, the waste from these farms is inadequately treated, resulting in the release of antibiotics into the wider environment.

In the face of such practices, it was inevitable that our soils and waters would be saturated with antibiotics. We have moved to a world where antibiotic pollution is a health hazard. But antibiotics are not quite in the same category as classical chemical pollutants: they rarely harm humans directly. So why does antibiotic pollution endanger us? The problem is evolution, which has led to the rapid dissemination of antibiotic resistance among previously susceptible bacterial populations, making these miracle drugs ineffective in many situations.

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he diversity of life forms we see on earth owes much to evolution, Charles Darwin’s epoch-defining theory. All forms of life—from single-celled bacteria to multi-celled humans, and not ignoring viruses that lie at the boundary of the living and the non-living—produce offspring that maintain genetic similarity with their parents, while also generating diversity. A pool of genetic variants can then compete for resources in a niche, and the principle of the “survival of the fittest” can operate, selecting for those variants that are best able to grow and reproduce in that niche. Genetics is more pronounced and better understood as a determinant of this competition in simple, single-celled organisms such as bacteria, rather than for mammals where competition among cells within a single animal or human operates is in a dichotomy with that among individuals.

How does evolution favour antibiotic resistance? Think of a pristine world where antibiotics never existed. We can imagine a population of frolicking, microscopic bacteria, twirling their outsize organs of motility, twisting and turning here and there, looking for food, growing, replicating their genetic material, and giving rise to little bacteria. They compete with each other when for example, nutrients are scarce, and those genetic variants that better utilise limited nutrients outperform their less fortunate compatriots. And then, the world is no longer what it used to be. We introduce antibiotics and the bacteria start dying. Many don’t know what hit them. A few bacteria heave a sigh of relief because genetic variation has given them the tools to evade or resist these killer molecules. They start proliferating, honing their genetic weaponry against antibiotics along the way, until the antibiotics humans enthusiastically used to commit genocide on our bacterial adversaries become useless. The resistant bacteria are here to stay, unless the development of resistance can be reversed.

That resistance would develop has always been a no-brainer. Darwin was done and gone well before antibiotics came. Bacterial resistance to penicillin had been discovered—in 1940—before its commercial production commenced. Some of the defining early work in bacterial genetics—performed in the 1940s—reported spontaneous evolution of antibiotic resistance by bacteria in the laboratory. But it was probably the scale and rate at which it developed, to the extent of being considered a serious threat to humankind, that took us by surprise.

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here are two broad ways by which a bacterium becomes resistant to an antibiotic. The first is the “every bacterial family on its own” method. A bacterium accidentally finds small variations in its genetic material, which enable it to evade death by antibiotic. These variations are inherited by its progeny. In other words, resistance is by vertical descent from parent to child. For example, Mycobacterium tuberculosis, which causes TB, becomes resistant to antibiotics predominantly in this way. The resistance-conferring variations are normally present in the bacterial protein with which the antibiotic interacts. They do not necessarily change the original function of the protein, just make it insensitive to the antibiotic. In general, molecular interactions in a living cell are highly specific, and small variations in one member of an interacting pair can result in large reductions in interaction affinities. In fact, drug designers often make new variants to evade known resistance mechanisms by making small changes to a pre-existing antibiotic structure.

The second mechanism is more devious, and could arm a bacterium with a resistance conferring protein very different from any protein the bacterium may already contain. Certain types of DNA are “mobilisable”, can move from one organism to another. One such DNA often discussed is called a “plasmid”. We often find that novel proteins that appear to be dedicated to conferring antibiotic resistance are encoded on such plasmids and other mobilisable elements. This is bad news.

Evolution is probabilistic, operating at the lower end of the scale. Finding a DNA sequence beneficial under a particular circumstance—amid the vast universe of possibilities—is clearly a low probability event. Hence, accidents and unfathomably large time-scales have played a non-negotiable role in creating the range of life we see today. In this light, one would assume that evolving a novel antibiotic evading molecule would be less likely than finding a needle in a haystack.

Imagine a situation where one bacterium, Anansi—of the 1000000000000000000000000000000 (one followed by thirty zeroes) individuals out there—accidentally finds such a molecule and gets it encoded onto a mobilisable DNA segment. The rest of the bacterial world—which may not have seen any molecule remotely resembling an antibiotic resistance-conferring protein—do not have to reinvent the wheel. They have to just receive this wisdom from Anansi, and once received spread the good news among their neighbours. As soon as it becomes apparent that the received wisdom is great, it spreads rapidly. And in an antibiotic-polluted world where a bacterium is likely to come across these killer molecules, these scraps from Anansi mean survival. Eventually, pretty much all bacteria in such a world will acquire this wisdom. Whereas the first mode of resistance spreads from parent to progeny thus limiting its prevalence, acquiring a mobilisable DNA element “horizontally” is more promiscuous, and on rare occasions may even occur across kingdoms of life.

It turns out, however, that for a bacterium, discovering a protein that confers antibiotic resistance is not as formidable a task as finding the needle in a haystack is for us. There are at least two reasons for this. The first is what Mariya Morar and Gerald Wright, in the 2010 issue of the Annual Review of Genetics, call the “genomic enzymology of antibiotic resistance”. This refers to the concept that many proteins that confer antibiotic resistance are not very different from proteins that perform essential cellular functions. In other words, a bacterium might harbour a protein necessary for its everyday function. Make a new copy of the gene that codes for this protein and give it a few minor tweaks. It not only retains the original life-giving version of the protein but acquires a new, mean machine that can brush antibiotics aside. This is clearly easier than finding an entirely new protein for resistance. It is not dissimilar to, say, a motorcycle manufacturer releasing newer versions of their flagship model with a differently tuned engine or with ABS, as opposed to inventing an entirely new class of motorcycle with black hole brakes or some such fancy addition. In the bacterial world, the class of proteins called beta-lactamases, which degrade penicillins into non-toxic products, are closely related to proteins normally involved in making and breaking the cell wall.

The second reason why bacteria may not be totally taken aback by the recent onslaught of antibiotics is the fact that antibiotics are derivatives of natural compounds that have been around in the environment for millions of years. Many antibiotics are in fact produced by soil bacteria as part of their natural lifestyles: presumably to aid them in their intense competition with other pesky neighbours. If a bacterium has to make antibiotics to kill off its competitors, it itself must first be resistant to the antibiotic it produces. If its resistance-conferring molecule was encoded on a mobilisable element, the creator becomes the destroyer: the bacterium might have given us a new antibiotic, but eventually double-crosses us by being Anansi to the rest of the bacterial world. And in that age-old arms race, its competitors would have also evolved resistance with or without Anansi; the original antibiotic producer would have evolved to make newer antibiotics without harming itself; its intended victims would have again evolved newer resistance mechanisms; and this ongoing cycle has been going on forever. Thus, natural antibiotic resistance is as old as the oldest antibiotics, which may have been known to humankind for a mere 100 years, but are probably a 100 million years old or more.

In short, antibiotic resistance was inevitable. To quote a 2010 article by Julian Davies, published in the Microbiology and Molecular Biology Reviews, “if antibiotic resistance is biochemically possible, it will occur”. We only have to blame ourselves for our negligence and complacence for having allowed it to rear its head to such an extent that we at times seem lost for ideas. So, what is the way forward? The World Health Organization came up with an “Antibiotic Action Plan” in 2015, which encourages infection prevention, prudent use of existing antibiotics, and the discovery and deployment of novel drugs.

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here are two types of things we should do to retain the edge over infections, besides public health measures such as sanitation and vaccination. One is at the policy level, to see how we can save the antibiotics we already have. It is clear that inappropriate use has resulted in antibiotic pollution, thereby encouraging the spread of resistance. Thus, effective regulations are essential to stop over-the-counter sale of antibiotics at pharmacies, which is rampant in the country.

The human gut is full of friendly bacteria, and antibiotics also kill these and at times cause diarrhoea. This could lead to resistance among these friendly bacteria; they could become Anansis, gifting resistance to an invading agent of a serious infection. In a 2013 article published in Frontiers in Microbiology, Miguel Gueimonde and colleagues point out that many bacteria we consume as probiotics often carry resistance determinants in mobilisable elements, and these are little short of time bombs.

One of the major causes of neonatal mortality in India is sepsis, a process by which certain molecules of our immune system are released into the bloodstream, resulting in tissue damage and often death. Such responses follow infection of the blood by errant bacteria, but not always. Such a condition is an emergency and the doctor does not have the time to determine whether the underlying cause was a bacterium or not. The doctor thus may prescribe—without the option of determining whether it is appropriate or not—an antibiotic, hoping that it would cure the child of the life threatening situation. The doctor appreciates the problem of antibiotic resistance and the contribution of inappropriate antibiotic use to its spread, but in the absence of alternatives is helpless. Thus, finding rapid diagnosis tools for identifying bacterial infections accurately is paramount to combating resistance.

Evolution is hard to fight except with a parallel evolution. Bacteria reinvent themselves to fight every new antibiotic. The only  is for us to maintain a steady flow of new antibiotics with novel targets.

The scale of this problem of resistance in bacterial sepsis is illustrated by a recent survey of nearly 90,000 infants across three large hospitals in Delhi, published in Lancet Global Health. This survey showed three varieties of bacteria—namely Acinetobacter, Klebsiella and Escherichia—responsible for most cases of sepsis in which a causative organism could be identified. Over 80 per cent of the Acinetobacters isolated in this study were resistant to more than three antibiotics. Somewhat reassuringly, the prospect of recovery for patients carrying multidrug resistant bacteria was not statistically worse than that for patients infected by sensitive bacteria. Acinetobacters are a class of rather robust bacteria, with some varieties resistant to all known standard antibiotics. They are a major nuisance not just in Delhi, but represent a global problem, often identified with infections acquired—primarily by weak individuals—in hospital, where the use of antibiotics eliminates most competition to these hardy bacteria.

Antibiotic resistance is not always infallible. It does come with an Achilles heel. In many cases, a bacterium resistant to an antibiotic may not be happy if the antibiotic is removed from its environment. If that happens, its relative, which lacks resistance, may soon start dominating the population. A prominent example relates to multi-drug resistant Acinetobacter and the last resort antibiotic colistin. In a 2013 report in Genome Research, Snitkin and coworkers used genome sequencing to show that while resistant populations of the pathogen emerged in response to the antibiotic, drug withdrawal quickly resulted in the dominance of the antibiotic sensitive variant.

Now, the observation that antibiotic resistance should be weak is not a given. Leading evolutionary biologists Diarmaid Hughes and Dan Andersson have shown that if bacteria live and multiply in amounts of antibiotic that are substantially less than lethal to the bacterial population, a new dynamic is established and resistance develops with no strings attached. In other words, unlike the colistin case above, in these situations, withdrawing the drug will not necessarily reverse resistance. We have observed this in our laboratory as well. In our natural environments, antibiotic concentrations are typically below lethal doses, and thus a barrier to the development of antibiotic resistance is effectively removed. More importantly, over-the-counter purchases of antibiotics and their use without a prescription almost certainly result in sub-optimally low concentrations of the antibiotic in our bodies, thus inviting trouble rather too close to home for comfort.

It is in this context that the negligence of some medical practitioners should be condemned. It has been brought to my attention that a Chennai doctor prescribed an anti-tuberculosis regimen to a patient without mentioning the dosage. This being a long-term treatment the patient—who is affluent and educated and cannot be classified as someone too ignorant to take a doctor’s advice lightly—ended up with suboptimal doses of antibiotic over a period of four months. Other, more careful doctors have intervened and corrected the situation. But one can always imagine a situation where the patient may never realise the gravity of the situation, resulting in a recipe for the evolution of—in the worst case—a seriously multidrug resistant Mycobacterium tuberculosis. If a doctor with the right credentials could do this, one can only imagine the situation in a country where, according to a recent survey by the World Health Organization, a majority of “doctors” are not medically qualified.

 

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t the end of the day, evolution is hard to fight except with a parallel evolution. Bacteria reinvent themselves to fight every new antibiotic we throw at them. The only way to overcome this problem is for us to reinvent ourselves and maintain a steady flow of new antibiotics with novel targets and newer mechanisms of action. Easier said than done. Developing a new drug is not easy. And there are always regulatory hurdles that may take years to jump over.

Bacteriophages are not a miracle cure. Bacteria do become resistant to bacteriophages, but bacteriophages then evolve to find a way around.

One possible solution, first proposed a hundred years ago, is a class of viruses called bacteriophages to fight bacteria. They were discovered as predators of bacteria by Frederick Twort and Felix d’Herelle in 1915 and 1917 respectively. The latter in particular proposed and showed that these viruses could be used to fight bacterial infections. Much earlier, a man called Hankin found an antibacterial agent in the Ganges, which many believe today were bacteriophages; but the evidence is not watertight.

In the early 1920s George Eliava set up a bacteriology institute in Tbilisi in Soviet Georgia. Having worked with d'Herelle in France and presumably influenced by his ideas, Eliava invited him to his institute in the 1930s, as part of Joseph Stalin’s call to the Soviets to cut the technology gap with Europe and the USA. d’Herelle worked there for a year and a half, and had his book on bacteriophage therapy translated into Russian and Georgian by Eliava and published in the Tbilisi National University. A potentially transformative long-term collaboration between Eliava and d’Herelle did not come to pass, in part because Eliava fell foul of the Soviet establishment and was executed. Nevertheless, bacteriophages continue to be used for bacterial infections to this day in Georgia. But it never took off in the West. Euphoria over the success of penicillin was one reason and d’Herelle did not help his cause by demonstrating the efficacy of bacteriophage therapy using incomplete experiments, which did not quite pass the thresholds for scientific rigour in the West. The Cold War was a further barrier in later decades. However, the problem of antibiotic resistance has led to a revival in phage therapy research.

As with most things, bacteriophages are not a miracle cure. Bacteriophages and bacteria are in conflict in nature and, both being capable of genetic variation, have maintained a healthy arms race over millions of years. That means bacteria do become resistant to bacteriophages, but bacteriophages then evolve to find a way around. In a clinical setting, it becomes important to see what treatment can result in bacterial resistance to a bacteriophage drug, and what situations do not. Can we for example generate a cocktail of bacteriophage variants to treat an infection where the spectre of resistance looms large? Maybe a combination of bacteriophages and antibiotics will work better. In fact, a combination of penicillin and a bacteriophage was shown to be more potent than either in combating various bacterial infections way back in the 1940s.

Another teething problem with bacteriophages is that unlike antibiotics, which can target a broad range of bacteria, bacteriophages are highly specific to one or a few types of bacteria. Therefore, accurate identification of the source of an infection would be required before treatment, calling once again for new, rapid, cost-effective diagnosis tools. One could presumably get around this problem by using a cocktail of carefully chosen combinations of bacteriophages.

Finally, bacteriophages, though predators of bacteria and not humans, do have molecular components that can activate an inappropriate and dangerous immune response from our bodies. But recent developments in genetic engineering should enable us to generate bacteriophage variants with reduced potency. Genetic engineering can also take us further: for example, it has enabled scientists to use bacteriophages as vehicles for delivering killer genes to their target bacteria, at least in laboratory settings.

These are of course early days, and it is time the basic sciences in bacteriology, virology and evolutionary biology came together with clinicians and drug developers to overcome the looming threat of antibiotic resistance—maybe “apocalypse”—by discovering newer (and re-discovering older) ways of fighting infections and also influencing policy and its implementation.