Issues Magazine

Microbes Making Their Mark in Drug Discovery

By Siouxsie Wiles

Living things that glow in the dark are showing great potential in the fight against drug-resistant tuberculosis.

For as long as I can remember I have been fascinated by how nasty microbes cause disease. How can something as tiny as a bacterium or virus kill something as complicated as us?

And kill us they do. It turns out that about one of every three people who die worldwide are killed by infectious microbes. That’s a staggering 14 million people each year. Even more frightening is the fact that, through the process of evolution, many microbes are becoming resistant to what treatments we do have.

Take the lung disease tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis. M. tuberculosis was discovered more than 100 years ago but, far from being eradicated, TB has turned into a worldwide disaster. M. tuberculosis is highly infectious, spreading from person to person through aerosols.

About one-third of the world’s population are believed to be infected with M. tuberculosis. Worldwide, there are 8.7 million new TB cases each year and three people die from TB every minute.

Although antibiotics are still effective for most TB patients, they have to be taken for 6 months to eradicate this wiley bacterium. Unfortunately, extensively antibiotic-resistant strains of M. tuberculosis now exist, and a staggering one in every 15 new TB cases worldwide is caused by drug-resistant strains. These cases can take 2 years to treat and cost 200 times more than regular TB, placing a huge burden on healthcare budgets. It is clear that there is a great and urgent need for new, effective antibiotics against this old foe.

Alongside nasty microbes, I am also fascinated by bioluminescence – the production of visible light by living creatures. This amazing phenomenon has evolved in a wide variety of organisms, with many different purposes. It allows fireflies to find a mate, anglerfish and glow worms to lure food, and nocturnal squid to camouflage themselves from predators.

The light produced by bioluminescent creatures is a by-product of a chemical reaction. The chemicals involved are known as luciferins and luciferases, and the reactions require oxygen and energy in the form of adenosine triphosphate (ATP). Luciferin and luciferase are just generic terms, and the chemicals themselves vary. At last count there were at least five different systems and plenty of bioluminescent organisms for which the luciferases/luciferins are still unknown.

One of my favourite bioluminescent creatures is a soil bacterium called Photorhabdus luminescens, which lives inside the gut of a minuscule worm. Together the deadly duo kill the larvae of many insects. The worms are eaten by the larvae and, once inside the larval gut, regurgitate their bacterial partner. Once regurgitated, the bacterium starts producing toxins, like the mcf (“makes caterpillars floppy”) toxin, which kill the larvae. P. luminescens also produces antibiotics (to stop other microbes growing in the larval carcass) and light (possibly to attract other larvae).

It’s said that during the US Civil War, soldiers with glowing wounds were more likely to survive than those whose wounds didn’t, a phenomenon they called “angel’s glow”. It’s likely the soldiers’ wounds contained P. luminescens, whose antibiotics would have killed off nastier microbes.

I have been very lucky to have made a career combining my twin passions of bioluminescence and infectious diseases. My team and I make nasty bacteria glow in the dark. We then apply our glowing bacteria to understand how various superbugs cause disease and to find new antibiotics to kill them.

Tagging bacteria with the genes for bioluminescence allows us to use light as a surrogate for physically counting the numbers of bacteria present in a sample, which is what we normally do after plating samples onto selective agar. Instead we can use a luminometer or charged coupled device (CCD) camera to give a measure of light intensity (called relative light units, or RLUs).

One of the first things we have to do is determine the relationship between light and bacterial numbers, but once this is done we can make a good estimate as to how many bacteria are present by using the RLUs. This can be especially useful if the bacteria take many weeks or months to grow on agar, as M. tuberculosis does.

Because only living bacteria glow, bioluminescence can also serve as an excellent marker for whether a particular treatment is working or not – if it is effective, the amount of light will decrease as bacteria are killed. With colleagues from the Auckland Cancer Society Research Centre, we are now using our glowing bacteria to find new drugs to treat TB.

With the need for more effective antibiotics and vaccines comes the need to use laboratory animals to test such treatments before they can be used in humans. Central to the use of animals in research is the promotion of the three Rs: replacement, reduction and refinement. If a non-animal alternative is available, it must be used. If not, scientists are compelled to obtain valid data using the fewest number of animals, at the same time minimising any pain or distress that those animals may experience. In this context, bioluminescent bacteria prove to be very useful.

Most people will have tried this simple experiment at home: if you hold a torch up against your hand you will see red light coming out the other side. This is because light can travel through flesh and skin. Sensitive CCD cameras that can detect very dim signals coming through flesh, a technique known as biophotonic imaging (BPI), allow researchers to visualise bioluminescent bacteria from inside a living mouse. Using BPI has allowed us to substantially reduce the number of animals we use in experiments. Where we would previously use five to eight animals at a given time for analysis of the number of bacteria present, we now routinely use five to eight animals per complete experiment, following the bioluminescent signal from each individual animal over time.

In addition to reducing animal usage, BPI also makes research more humane. For example, the rapid and uncontrolled expansion of many nasty bacteria can rapidly kill their host – this is true in people and mice. We use BPI as a guide to how many bacteria are present, and routinely perform humane euthanasia before the animals show any sign of disease.

Who would have thought that fireflies and other glowing creatures would be so important in the fight against superbugs?

Siouxsie can be found blogging and podcasting, as well as working with graphic artist Luke Harris and his team on a series of animations to tell the tales of some of nature’s amazing glowing creatures and the myriad uses of bioluminescence in science at http://youtu.be/kP_RaHo1Pmw