The race against antibiotics resistance Antibiotics and resistance traits co-evolved over several hundred million years, shaping the evolution and ecology of microbes and fungi. Within less than a century of using and over-using antibiotics, we have managed to spread resistance genes so far and wide that we are now facing the threat of a dramatic loss of the protection that they have provided until now. Michael Gross reports. Fungi and bacteria have coexisted and competed for more than a billion years, so it is no wonder that they have evolved a whole arsenal of weapons against each other. In its bid to fight bacterial infections, medicine has adopted and modified at first fungal and then also bacterial antibiotics, starting with penicillin, which was discovered less than a century ago, followed by others like tetracycline and erythromycin. In a recent phylogenetic paper, the group of Gerard Wright at McMaster University in Hamilton, Canada, provides an impressive illustration of the different timescales at work (Nat. Microbiol. (2019) https://doi. org/10.1038/s41564-019-0531-5). Actinobacteria are soil bacteria but in many ways act like fungi, in that they form mycelia and some can live in symbiosis with plant roots. They also produce glycopeptide antibiotics, such as vancomycin and teicoplanin, which include a peptide moiety that is assembled by enzymes rather than by ribosomes. The relatively large group of genes required for this synthesis has facilitated the phylogenetic analysis at the group level, even though individual genes may disappear or be replaced. The results suggest that the functional antibiotic and the resistance genes that actinobacteria need to protect themselves go back at least 150 million years, possibly even 400 million years. By contrast, the history of its use in medicine is rather shorter. Science discovered vancomycin in the 1950s. It entered into significant clinical use after the emergence of methicillin-resistant Staphylococcus aureus (MRSA) in the late 1970s. Within a decade after its introduction, clinical resistance was observed in pathogenic enterococci. Many other antibiotics introduced in the second half of the 20th century have suffered the same fate — resistance
spread faster than new antibiotics could be introduced. The problem is exacerbated by careless use of antibiotics, including its application for non-medical reasons in agriculture (Curr. Biol. (2013) 23, R1063–R1065). There is a growing concern that medicine is losing the race and we may be approaching a post-antibiotics age. Spread of resistance The World Health Organisation has highlighted the imminent global threat of antibiotics resistance in regular reports. It has warned that, by 2050, the global death toll from antibioticresistant strains of pathogens may be 10 million people per year. While the emergence of hospital ‘superbugs’ such as MRSA in the modern and perfectly equipped health institutions of the Western world has
found a big media echo, even larger problems are emerging in poorer countries in the tropics, where infectious diseases are still a big public health issue and the spread of resistance may not even be recorded in detail. The group of Jesse Goodman at the Georgetown University Medical Center in Washington, DC, USA, has compiled a meta-analysis of reports regarding resistance to two groups of antibiotics, the carbapenem group and the polymyxins, including colistin. Carbapenems were introduced as reserve antibiotics to treat severe infections displaying resistance to commonly used antibiotics. However, as their use increased, resistance genes also spread, leaving polymyxins as the treatment of last resort. Goodman’s group now presents data of the spread of resistance against either or both of these antibiotics in South-East Asia, where there is a very real danger that areas where both resistances overlap will have to deal with untreatable bacterial infections (Int. J. Antimicrob. Ag. (2019) https://doi.org/10.1016/j. ijantimicag.2019.07.019). There is also a lack of official data, as only three of the 11 countries in this area have filed data on these resistance problems for the most recent WHO reports.
Super bug: Methicillin-resistant Staphylococcus aureus (MRSA), shown here with a dead human blood cell, has become a widely feared ‘hospital superbug’ resistant to multiple drugs. Widespread use and misuse of antibiotics has, however, produced many other multidrug-resistant strains. (Photo: NIAID/Flickr (CC BY 2.0).)
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Magazine The study found that carbapenemresistant strains of Escherichia coli were documented in eight of these countries, and resistant Klebsiella strains even in nine of them. Polymyxin resistance was documented in eight countries. The geographic distributions of both groups of antibiotics were found to be overlapping in all of these eight countries. “The picture the data paints is of a serious emerging public health threat. We document that resistance to each drug is geographically widespread in the region, including many areas where the distribution of strains resistant to each type of antibiotic overlaps,” Goodman says. “Although combined carbapenem and colistin resistance is fortunately still rare, the coexistence of mobile resistance genes for both drugs in the same areas, such as we describe, raises the risk of organisms acquiring both, causing essentially untreatable infections.” Finding new antibiotics Discoveries of new antibiotics are few and far between. Earlier this year, the group of Ramiz Boulos at Flinders University, Australia, reported a novel compound proven to be effective in rats against the notorious Clostridium difficile infection (Sci. Rep. (2019) 9, 158). C. difficile infections in the large intestine are often observed in patients whose gut microbiome is compromised, for instance after long treatment with antibiotics. “Ramizol is an extremely welltolerated antibiotic in rats, with good microbiology and antioxidant properties,” the authors conclude. “Furthermore, Ramizol has high chemical stability and a scalable and low cost of manufacturing. These advantages might position it favourably in a CDI market with few treatment options.” Boulos is CEO of Boulos & Cooper Pharmaceuticals, which will likely pursue this further. Other pharmaceutical companies aren’t always keen on investing in the development of new antibiotics, as the short time window of use before resistance arises, combined with the expectation of affordable pricing, especially in the developing world where infectious diseases are more widespread, could strain the profitability of such endeavours. A report released in January by DRIVE-AB (http://drive-ab.eu/), an R860
international consortium managed by the University of Geneva and AstraZeneca, has suggested that a market entry reward of $1 billion per antibiotic globally could significantly increase the number of new antibiotics coming to the market in the next 30 years. The cost of developing a new antibiotic has been estimated at $2.7 billion. Francesco Ciabuschi from Uppsala University explained: “our simulations predict that introducing market entry rewards could potentially help to bring to market a total of 16 to 20 new truly innovative antibiotics in the next 30 years. Without incentives, some scientifically promising treatments would probably never make it to patients.” The report aiming to create a more favourable financial ‘ecosystem’ for the development of new antibiotics envisages that the G20 summit of nations should take the initiative to raise such a reward and also to ensure that it is linked to the conditions of sustainable use and affordable access to the new drug. Where the market fails to provide sufficient incentives, non-profit organisations may step in. In the fight against tuberculosis, for instance, multidrug resistance is a major threat and profitability of drug development is low. The non-profit TB Alliance, founded in 2000 after a meeting in Cape Town, South Africa, works to bridge the gap. In August, it could celebrate a major success winning FDA approval for its new antibiotic pretomanid, which in combination with two existing drugs achieves recovery rates that are three times better than previously available treatments. Beating bacteria In academic laboratories, researchers are also working on new ways of outwitting the bacterial defences against existing antibiotics. For instance, the group of Gad Frankel at Imperial College London, UK, has investigated the ways in which Klebsiella pneumoniae, a common cause of lung and wound infections in hospitals, becomes resistant to carbapenem (Nat. Commun. (2019) 10, 3957). By analysing the crystal structure of the OmpK36 pore protein of a resistant mutant, they found that a mutation narrowing the channel makes K. pneumoniae resistant to carbapenems and other drugs that
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have to enter the bacterial cells to become effective. For the bacteria, this adaptation comes at a significant fitness cost, as it also restricts uptake of nutrients, such as lactose. This finding explains why the resistance trait is only found in environments such as hospitals where antibiotic use is prevalent. Knowing that the narrower pore blocks the entrance of antibiotics leads to clear treatment strategies. Treatment with antibiotics has to be combined with a measure to ensure their entry into the bacterial cells. As Frankel puts it: “we hope that it will be possible to design drugs that can pick the lock of the door, and our data provides information to help scientists and pharmaceutical companies make these new agents a reality.” Another way of making the best possible use of existing antibiotics is to improve and speed up the decision of which drug or combination of drugs to use. It is already common practice that a physician prescribes a first-line antibiotic that is widely used and, when the infection turns out to be resistant to that, falls back on a second-line option that is held in reserve. Precisely because they are used less frequently, these second-line drugs are less likely to encounter resistance. This sequential treatment may often be little more than a combination of chance events, where the physician improves the likelihood of success by rolling the dice again. This carries a risk of dangerous failure, however, when both treatments remain ineffective and the infection continues to spread. A more reliably successful approach could be developed on the basis of knowing how likely certain combinations of drugs are to succeed based on so-called collateral sensitivities. Thus, if two antibiotics have very different mechanisms of action, a strain evolved to be resistant to one may be more sensitive to the other. To explore the possibilities of using collateral sensitivities in practice, computer scientist Daniel Nichol at the University of Oxford, UK, working with biomedical experimentalists in the USA, combined mathematical modelling with in vitro evolution in E. coli to test how a pre-existing resistance to the antibiotic cefotaxime influences the response towards other drugs and the possible evolution of resistance (Nat. Commun. (2019) 10, 334).
Magazine The tests showed that both collateral sensitivity and cross-resistance can evolve in strains exposed to a sequence of two drugs with a certain probability. Assessing the probabilities for all reasonable combinations of drugs will be necessary to identify which offer the best opportunities to overcome resistance with the agents already available. Future antibiotics While combination and supplementation of existing antibiotics may help to expand the lifespan of their protection, nobody denies that there is still the need to develop more new antibiotics, and particularly ones with fundamentally new modes of action that can bypass existing resistance mechanisms. For this endeavour, the phylogenetic work on glycopeptide antibiotics from Gerard Wright’s group mentioned in the introduction can also be helpful. Based on the large amount of sequence data now available, Wright says, “we can begin to find long lost ‘cousins’ [of the known glycopeptide antibiotics]. That is, biosynthetic gene clusters that retain some of the family’s key characteristics, but not all, or ones that have extra genes and thus structural attributes.” Studying these strains, the researchers hope to find related compounds that may have antibiotic activity based on different modes of action. “For example, in work that is currently under review, we identified biosynthetic gene clusters that were very similar to glycopeptide antibiotics, but they were clearly different,” Wright explains. These differences included longer predicted peptide sequences and different sugars, or even the absence of sugars. “We then were able to purify the associated compounds and found that they are indeed antibiotics and that they operate by a different mechanism than glycopeptides. This strategy of using the phylogeny to narrow down on a region of bioactive compound space is I think generally applicable to other kinds of compounds,” Wright concludes. Thus, although a dark future with the return of untreatable bacterial infections has become a distinct possibility, research can still offer a few silver linings. Michael Gross is a science writer based at Oxford. He can be contacted via his web page at www.michaelgross.co.uk
Amber David A. Grimaldi What is amber? Amber is one of Earth’s most intriguing and beautiful substances, which largely functioned as liquid band-aid for trees. Amber is fossilized resin, produced by various plants in response to physical injury, by boring insects or by branches snapped off and trunks gashed open. Resin effectively seals a plant wound to prevent invasion by fungi, bacteria or insects, though some insects actually use the resin — stingless bees, for example, gather resin to build their nests. What is amber made of? Unlike gums and saps, which are water soluble, resins generally are polymers of diand sesquiterpenes. Terpenes are multicyclic hydrocarbons and comprise some 60% of plant biomolecules (think: menthol, clove oil, cannabinoids and many others). The polymers crosslink (either quickly or gradually over thousands to millions of years) to form amber, which is hard and virtually inert, which is why it can’t be dissolved or melted. But some amber hardly crosslinks at all and can be dissolved, particularly the dammartype amber from India and southeast Asia. Sometimes the chemistry is perplexing. Years ago I sent 50 millionyear-old samples from New Jersey to chemist colleagues for analysis, who thought I had discovered the oldest fossil landfill. The material was a hard, dense polystyrene, and we later learned that some trees like witch-hazels (Hamamelis spp.) produce a resin with natural styrene. When does resin become amber? An arbitrary age has been established at 40,000 years old, which is the limit of 14C dating. Younger than that the substance is often called ‘subfossil resin’ or ‘copal’. If the molecular composition of amber has hardly changed, is it still a fossil? Yes. First, there is usually some molecular change in amber from the original resin, in the loss of radicals and highly volatile components, in
cross-linking and polymerization. Second, a fossil is usually defined as the remains of an organism that are of significant age and that were naturally buried or preserved; the remains do not have to be petrified or mineralized, nor even be an extinct species. A mammoth frozen in permafrost is a fossil. The insects and other life forms preserved as inclusions in amber (Figure 1) may have life-like fidelity, even down to subcellular structures, but they are only fossilized facsimiles of the original organism, as their molecular composition has largely been degraded. Where is amber found and how old is it? Amber is found worldwide, from the arctic to Antarctica, the oldest formed by tree ferns, about 320 million-years ago (mya) during the Carboniferous. Trace quantities of fossil resin occur throughout terrestrial deposits, but lumps of amber were for some reason not produced until the Early Cretaceous, approximately 130 mya, even though conifer trees were abundant before. Geological periods of greatest amber production seem correlated with particularly wet paleoclimates, in the Late Triassic, the Early to midCretaceous and the Eocene. The Jurassic was quite dry, which may explain the paucity of fossil resins from that period. By the way, new deposits are being discovered on a yearly basis, and those worked for centuries are still yielding remarkable discoveries. Where are the largest deposits? Baltic amber (38–45 million-years old) is by far the world’s largest deposit. It has been mined over the centuries and over 3,000 fossil species (mainly arthropods) are preserved in it. Other large deposits are found in northern Myanmar (~100 million-years old), the Dominican Republic and Chiapas Mexico (15–17 million-years old). Massive quantities of Tertiary dammar-type amber are preserved in India (Figure 1) and Indonesia. Smaller but scientifically invaluable deposits occur from the Cretaceous, in Lebanon (130–115 mya), Spain (105 mya), France (100 mya), New Jersey (90 mya), and western Canada (72 mya). Formation of an amber mother lode requires a natural disaster involving a forest of trees that oozes great stalactites of resin, concentration of the hardened resin (generally by water
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