I saw a paper in Science this week that was simultaneously very exciting and, frankly, terrifying. It involved the study of antibiotic resistance in Escherichia coli. Many studies have been done looking at how E. coli and other bacteria can evolve resistance to both single and multi-drug environment. These are normally conducted in flasks of liquid culture, though, where the bacteria grow freely. This study was different as it included a spatial dimension to the experiment, something that bacteria would have to overcome in the real world as they go about their business.
The researchers made a huge petri dish more than a metre long. The plate was divided into 9 zones, at either end there was a zone with just agar where the E. coli could grow at their leisure, the next zone in was laced with antibiotics, ciprofloxacin, at a level that was just above what the bacteria could tolerate. The next zone had ten times that concentration, the next 100 times and the final central zone had 1000 times the initial concentration.
Below is a short video of the set up with a time lapse of how the bacteria were able to colonise even the 1000x antibiotic concentration area in just 11 days.
Do you see what I mean when I say exciting and terrifying? It is a simple and elegant set up that beautifully demonstrates evolution by natural selection before your very eyes. It also shows the relentless forward march of bacteria as they take only a week to evolve the ability to set up shop in an place that was previously completely lethal to them.
In another set up the researchers didn’t have the intermediate steps but went straight from no antibiotics to a massive dose, in this circumstance no bacteria were able to adapt. They were able to show, then, that having a gradation helped the bacteria along the way, a feature that hospitals might be able to use to their advantage in their fight against microbes.
Another interesting discovery was that it wasn’t always the bacteria that had the greatest resistance to the antibiotic that went on to dominate the next zone. Sometimes a bacterium that was less resistant but was able to replicate itself faster than a more highly resistant neighbour was able to gain the upper hand and outcompete that neighbour. Resistance, then, is not the only factor we need to look at.
If you’ll allow me a bit more depth on the genetics it appears that the bacteria had two main strategies. The researchers were able to take samples from the agar plate and sequence the genomes of the bacteria thereby allowing us to see how the colonies had evolved over the 11 days. Having compared the mutants to the original wildtype colony they found that they tended to sort themselves into two distinct populations: some bacteria had lots of new mutations (>60) whilst others had very few (<12).
In those bacteria with lots of mutations they found that several of them had occurred in the dnaQ gene. All organisms, even little bacteria, have proof reading mechanisms that check for mistakes when DNA is replicated in preparation for a cell to divide. These are important, they protect us from damaging mutations that inhibit the function of a gene we need or perhaps confer a new function, like immortality, in the example of cancer. dnaQ is the gene that encodes Polymerase III which is vital to proofreading in E. coli during DNA replication.
Once the proofreading mechanism is disrupted this allows even more mutations to start springing up. It’s as if the bacteria has sabotaged its own genetic machinery to kickstart the evolutionary process. A very risky strategy for the individual but great for the species.
Although these colonies had more mutations most of them were what we call synonymous. This means that the nucleotide changed but, due to the redundancy of the genetic code, the amino acid was not altered. This means that the mutation is effectively silent and protein function continues unimpeded.
The other group of E. coli, with very few mutations, had not sabotaged their proofreading mechanisms and so had fewer mutations, both good and bad. In this population the mutations that had made it through were generally nonsynonymous, i.e. they were changing the amino acid sequence in the protein and were therefore more likely to be having an effect on protein function. Whilst this second group, then, had fewer mutations they were each more likely to have a more significant impact.
The authors explain that the MEGA-plate (microbial evolution and growth arena-plate), as they call it, is not designed to mimic a real world environment but it certainly can be useful in knowing what characteristics are key in driving evolution. They end on a point that I rather like. Given the large scale and ease of construction of the MEGA-plate it could be a useful tool in science education and outreach. I’m always pleased when I see scientists thinking of ways to engage with the general public and it’s particularly important when the topic is one as important as antibiotic resistance.
One of the key arenas for fighting antibiotic resistance is not just the hospital ward or the MEGA-plate but in the consciousness of the public. We have to get them out of the habit of asking for antibiotics when they have a cold caused by a virus. We have to empower GPs and primary care physicians to be able to send them packing if that’s what they insist on. We have to make sure patients take the full course and don’t just stop taking their medicine once they feel better. We have to tackle the problem of antibiotics being available by the pill over the counter in developing countries by making sure that as much of the world as possible has access to cheap and effective healthcare. We have to stop using antibiotics as standard in animal husbandry as growth promoters.
This is a war we are fighting on many fronts, but it is a war we must win. I don’t much like the idea of dying just because I scratched my finger and got some dirt in it. I don’t fancy having surgery without being pumped full of antibiotics to make sure I don’t get infected. And I really wouldn’t want to have to be immunosuppressed for an organ donation or to fight cancer without having a barrage of antibiotics to help me stay healthy. Understanding how these bacteria evolve, then, is extremely important work and, the next time you have a bit of a sniffle, don’t go asking your doctor for antibiotics. Think of the bigger picture.