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Antibiotic resistance is ancient: implications for drugdiscovery

Gerard D. Wright1

and Hendrik Poinar2

1M.G. DeGroote Institute for Infectious Disease Research, Department of Biochemistry and Biomedical Sciences, McMaster

University, 1280 Main St W., Hamilton, ON, L8S 4K1, Canada2McMaster 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 antibio-

tics, denotes the importance of taking microbial chemi-

cal ecology and deep metagenomic profiling intoaccount in the development and use of antibiotics.

Antibiotics represent one of our most effective therapeutic

defenses against infectious diseases. Despite this resound-

ing success, our continued use of antibiotics is under

enormous threat. This is because of two parallel chal-

lenges. The first 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 finite efficacy and lifetimes as resistance

always emerges. Resistance to the first antibiotics includ-

ing

penicillin

and

streptomycin

was

quickly

reported

fol-lowing their discovery [2]. These results suggested a

paradoxical population of pre-existing resistant organ-

isms, 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 resis-

tance 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 circu-

lating

vertically

and

horizontally

throughout

microbialcommunities. For example, by 1998, there were over 75

b-lactamases that shared

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 first use of

penicillin in the 1940s. Indeed, phylogenetic analyses of

disparate but similar proteins related to b-lactamases

suggest an ancient root [4].

Recently, we provided the first 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

theYukon.Usinga seriesof optimizedPCRassays coupled

with high throughput sequencing, we identified DNA

segments stemming from floral (grasses and willow) and

faunal (mammoth, bison and horse) remains characteris-tic of anArctic Pleistocene assemblage.Theabsenceofkey

Holocene species, such as moose, elk and spruce, con-

firmed 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 fluorescent 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 bacte-

rial species to ascertain that the qualitative bacterial

content of these sediment cores were similar and consis-

tent with modern temperate soils. Themetagenomic DNA

we

sampled

were

Pleistocene

era

in origin and reflected anormal microbial soil environment. We then used probes

for several antibiotic resistance elements and unambigu-

ously identified markers ofb-lactam (b-lactamase) and

tetracycline (ribosomal protection) resistance, as well as

genes responsible for resistance to the glycopeptide anti-

biotic vancomycin. The latterwas especially intriguing as

it requires the expression of at least three gene products

that together act to reprogram the chemical structure of

peptidoglycan, themolecular target ofvancomycin. One of

these ancient genes, a variant of vanA that encodes an

ATP-dependent D-alanyl-D-lactate ligase, was synthe-

sized, recombinantly expressed and the purified protein

confirmed to have the expected biochemical activity. Fur-

thermore, 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

analysisconfirmedthat thegeneswerebonafideantibiotic

resistance elements.

Whydoes resistancepredate theantibiotic 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

Forum: Science & Society

Corresponding author:Wright, G.D. (wrightge@mcmaster.ca).

Keywords: antibiotic; ancient DNA; vancomycin; beta-lactamase; tetracycline;

aminoglycoside.

157

mailto:wrightge@mcmaster.camailto:wrightge@mcmaster.ca

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resistance mechanisms and these have clearly been mobi-

lized horizontally through microbial populations in addi-

tion 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 specific efflux and highly

selective

influx

systems.

The

end

result

is

that

most

bac-teria 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

reflected in the paucity ofnew antibiotic clinical candidates

coming to market [1]. It is increasingly difficult to identify

new antibiotic leads, a situation that is resulting in a large

unmet clinical need.

Why is it so hard to find 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 ofdrugs. However, given that the resistome is so broad and

ancient, the presence of highly evolved and efficient resis-

tance 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 hy-

drolysis) is a reflection 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 aminogly-

coside antibiotics can occur by mutation of the target

ribosome,

via

methylation

of

the

ribosomes

by

a

cadre

ofhighly efficient enzymes, by blockade of compound entry

into the cell, efflux out of the cell and at least three distinct

enzyme families (kinases, acetyltransferases and nucleo-

tidyltransferases), each with dozens of members that can

chemically modify the drugs [10].

Faced with this extensive diversity of resistance al-

ready hardwired into the microbial pan-genome, it is

reasonable to assume that natural products are poor

choices for newdrug leads. Rather, totally syntheticmole-

cules should offer chemical scaffolds that are not suscep-

tible 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 campaignsover the past two decades inmuchof

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

significant disadvantage in antibacterial drug discovery.

The reason for this poor record may lie in the selection of

chemical scaffolds that populate the libraries ofmostlarge

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

efflux proteins, which have evolved to maintain chemical

homeostasis with the environment.

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 offerprivileged structures that target vital

bacterial

targets. Careful exploration of such molecules

(in-cludingmanythat werediscardedasdrug leadsover thepast

decades) to identify theirmolecular targetsand 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 adhoc man-

ner).A robustpreclinicalplatformwhere leadcompoundsare

systematicallyprobed fortheirmicrobialvulnerabilitywould

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 andring size along with creating more rigid compounds) may

result in chemical libraries with more affinity for microbial

targets and the ability to penetrate cells and avoid efflux.

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. Com-

pounds that have weak or non-obvious antimicrobial ac-

tivity 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

newantibiotic 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.

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doi:10.1016/j.tim.2012.01.002 Trends in Microbiology, April 2012, Vol. 20, No. 4

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