Forum: Science & Society

Antibiotic resistance is ancient: implications for drug discovery

Gerard D. Wright1 and Hendrik Poinar2
1

M.G. DeGroote Institute for Infectious Disease Research, Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main St W., Hamilton, ON, L8S 4K1, Canada 2 McMaster Ancient DNA Center, Department of Anthropology, McMaster University, Hamilton, ON, L8S 4L9, Canada

An unfailing observation over the past 70 years is that resistance to all antibiotics emerges eventually after use in the clinic. Where does this resistance come from? Recent work has shown that antibiotic resistance genes are common in metagenomes of ancient sediments. This prevalence of resistance, well before the use of antibiotics, denotes the importance of taking microbial chemical ecology and deep metagenomic proling into account in the development and use of antibiotics. Antibiotics represent one of our most effective therapeutic defenses against infectious diseases. Despite this resounding success, our continued use of antibiotics is under enormous threat. This is because of two parallel challenges. The rst is diminishing interest and investment in new antibiotic drug discovery by the pharmaceutical sector [1]. The second is bacterial resistance to antibiotics. All antibiotic drugs introduced into the clinic have proven to have nite efcacy and lifetimes as resistance always emerges. Resistance to the rst antibiotics including penicillin and streptomycin was quickly reported following their discovery [2]. These results suggested a paradoxical population of pre-existing resistant organisms, even in the absence of the evolutionary pressure exerted by the drugs. The discovery in the mid-1950s of transferable resistance ushered in a greater appreciation of gene mobilization in the spread of antibiotic resistance and implied an even deeper reservoir of antibiotic resistance genes. The cloning and sequencing of antibiotic resistance genes revealed multiple genes belonging to large families as the genetic causes of resistance. This genetic diversity was again consistent with a large reservoir of genes circulating vertically and horizontally throughout microbial communities. For example, by 1998, there were over 75 b-lactamases that shared < 95% amino acid sequence similarity (in addition to the dozens of sequences with > 95% homology), pointing to a remarkably large number of distinct genes and proteins with anti-b-lactam antibiotic activity [3]. It was highly unlikely that such extensive genetic diversity could have arisen since the rst use of penicillin in the 1940s. Indeed, phylogenetic analyses of disparate but similar proteins related to b-lactamases suggest an ancient root [4].
Corresponding author: Wright, G.D. (wrightge@mcmaster.ca). Keywords: antibiotic; ancient DNA; vancomycin; beta-lactamase; tetracycline; aminoglycoside.

Recently, we provided the rst direct molecular evidence for antibiotic resistance in ancient sediment samples [5]. In this work we extracted total DNA from 30 000-year old permafrost cores from a well-dated site in the Yukon. Using a series of optimized PCR assays coupled with high throughput sequencing, we identied DNA segments stemming from oral (grasses and willow) and faunal (mammoth, bison and horse) remains characteristic of an Arctic Pleistocene assemblage. The absence of key Holocene species, such as moose, elk and spruce, conrmed that the samples were ancient and predated the modern antibiotic era by several millennia. To control for bacterial contamination in the center of cores, we spiked all equipment with a green uorescent protein-producing strain of Escherichia coli and monitored its movement (leaching) induced by the sampling procedure. Next we designed 16S rDNA probes to amplify and identify bacterial species to ascertain that the qualitative bacterial content of these sediment cores were similar and consistent with modern temperate soils. The metagenomic DNA we sampled were Pleistocene era in origin and reected a normal microbial soil environment. We then used probes for several antibiotic resistance elements and unambiguously identied markers of b-lactam (b-lactamase) and tetracycline (ribosomal protection) resistance, as well as genes responsible for resistance to the glycopeptide antibiotic vancomycin. The latter was especially intriguing as it requires the expression of at least three gene products that together act to reprogram the chemical structure of peptidoglycan, the molecular target of vancomycin. One of these ancient genes, a variant of vanA that encodes an ATP-dependent D-alanyl-D-lactate ligase, was synthesized, recombinantly expressed and the puried protein conrmed to have the expected biochemical activity. Furthermore, we also determined the three-dimensional structure of the enzyme and found that it was essentially indistinguishable from modern VanA ligases that are associated with vancomycin resistance in the clinic. This analysis conrmed that the genes were bona de antibiotic resistance elements. Why does resistance predate the antibiotic era? Bacteria originated over 3.8 billion years ago and based on the genetic divergence of antibiotic biosynthetic gene clusters, antibiotics are at least hundreds of millions of years old [6]. Bacteria therefore have been exposed directly or indirectly to antibiotics and their derivatives for an equal period of time. Antibiotic producers must co-evolve self-protective
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resistance mechanisms and these have clearly been mobilized horizontally through microbial populations in addition to being evolved independently. Furthermore, bacteria are exposed to bioactive molecules produced by fungi, plants and many other organisms and have developed a highly sophisticated series of countermeasures to avoid toxic compounds including broad specic efux and highly selective inux systems. The end result is that most bacteria are resistant to antibiotics [7]. This observation is distilled in the concept of the antibiotic resistome [8,9] the collection of all genes that directly, or indirectly, contribute to antibiotic resistance in microbes. The impact of the resistome on drug discovery is reected in the paucity of new antibiotic clinical candidates coming to market [1]. It is increasingly difcult to identify new antibiotic leads, a situation that is resulting in a large unmet clinical need. Why is it so hard to nd new antibiotics? Part of the answer lies in the chemical matter exploited in drug discovery. The majority of antibiotics in current use are natural products or their derivatives and microbial natural products in particular have been outstanding sources of drugs. However, given that the resistome is so broad and ancient, the presence of highly evolved and efcient resistance mechanisms to these antibiotics is not unexpected. Indeed, most natural product antibiotics are targeted by several resistance gene products. For example, the number and diversity of b-lactamases noted above (including two highly distinct chemical mechanisms of lactam-bond hydrolysis) is a reection of the fact that b-lactam antibiotic production is not uncommon in environmental microbes and that they have been producing these compounds for hundreds of millions of years. Resistance to the aminoglycoside antibiotics can occur by mutation of the target ribosome, via methylation of the ribosomes by a cadre of highly efcient enzymes, by blockade of compound entry into the cell, efux out of the cell and at least three distinct enzyme families (kinases, acetyltransferases and nucleotidyltransferases), each with dozens of members that can chemically modify the drugs [10]. Faced with this extensive diversity of resistance already hardwired into the microbial pan-genome, it is reasonable to assume that natural products are poor choices for new drug leads. Rather, totally synthetic molecules should offer chemical scaffolds that are not susceptible to pre-existing resistance. In fact, this was a consideration for the abandonment of natural product leads for libraries of synthetic compounds in antibiotic discovery campaigns over the past two decades in much of the pharmaceutical sector. However, as evidenced by the complete absence of new synthetic antibiotic drug leads during this time [11], such compounds seem to be at a signicant disadvantage in antibacterial drug discovery. The reason for this poor record may lie in the selection of chemical scaffolds that populate the libraries of most large pharmaceutical companies, which are not optimized for bacterial transport but rather for human disease targets. As a result, compounds either fail to penetrate bacterial cells or are substrates for the great number of bacterial efux proteins, which have evolved to maintain chemical homeostasis with the environment.
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The recognition of the ancient and modern antibiotic resistomes offers several solutions to the current stalemate in antibiotic drug discovery. First, while bacteria have evolved over hundreds of millions of years to detoxify small molecules, at the same time, organisms have co-evolved molecules with direct and indirect antibiotic action. These natural products offer privileged structures that target vital bacterial targets. Careful exploration of such molecules (including many that were discarded as drug leads over the past decades) to identify their molecular targets and a systematic search for pre-existing countermeasures in environmental bacteria can guide the development of semisynthetic derivatives that can evade many resistance mechanisms. Indeed, this approach has proven to be successful in the development of several generations of antibiotics over the past decades (although generally applied in an ad hoc manner). A robust preclinical platform where lead compounds are systematically probed for their microbial vulnerability would result in more methodically triaged leads. Second, the elaboration of synthetic molecules to more closely mimic natural products (e.g. increasing the number of chiral centers, hydrogen bond donors or acceptors and ring size along with creating more rigid compounds) may result in chemical libraries with more afnity for microbial targets and the ability to penetrate cells and avoid efux. Approaches such as diversity oriented synthesis [12] offer routes to such compounds. The third solution is the use of combinations of bioactive compounds to effectively increase chemical space. Compounds that have weak or non-obvious antimicrobial activity can, when combined, uncover cryptic synergy resulting in enhanced antibiotic activity [13,14]. Such combinations mimic natural product production in microbes [15] that have evolved over millennia. These examples are only a few of the options for new antibiotic drug discovery that are informed by our growing understanding of the antibiotic resistome and its ancient origins. The words of Winston Churchill are a guide for a new era in antibiotic discovery: The farther backward you can look, the farther forward you are likely to see.
References
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12 Galloway, W.R. et al. (2009) The discovery of antibacterial agents using diversity-oriented synthesis. Chem. Commun. (Camb.) 18, 24462462 13 Ejim, L. et al. (2011) Combinations of antibiotics and nonantibiotic drugs enhance antimicrobial efcacy. Nat. Chem. Biol. 7, 348350 14 Spitzer, M. et al. (2011) Cross-species discovery of syncretic drug combinations that potentiate the antifungal uconazole. Mol. Syst. Biol. 7, 499

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15 Challis, G.L. and Hopwood, D.A. (2003) Synergy and contingency as driving forces for the evolution of multiple secondary metabolite production by Streptomyces species. Proc. Natl. Acad. Sci. U.S.A. 100 (Suppl. 2), 1455514561
0966-842X/$ see front matter 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2012.01.002 Trends in Microbiology, April 2012, Vol. 20, No. 4

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