Australasian Science Magazine

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By Brigitta Kurenbach & Jack Heinemann

The overuse of antibiotics has led to a dramatic rise in the number of untreatable infections. To make matters worse, other chemicals like weed-killers can reduce the susceptibility of bacteria to antibiotics.

In many parts of the world, treatable bacterial diseases are becoming untreatable because they are resistant to many of the antibiotics we used to take. The most prominent example for this is tuberculosis, for which drug-resistant varieties have been confirmed and are spreading.

This is not a problem restricted to developing countries with inadequate medical capacities. Infections with antibiotic-resistant organisms forming the so-called ESKAPE group (named after the members Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii,

Pseudomonas aeruginosa and Enterobacter spp.) are increasing in hospitals around the world. If patients become infected, treatments take longer, are more expensive, may require the use of drugs that are more toxic to humans, or in the worst cases fail altogether.

The outlook isn’t good. We are heading towards a post-antibiotic future where infections from a small cut can be deadly or where routine surgery may be deemed too risky because the probability of infections with one of these multi-resistant pathogens is high.

The rise of antibiotic resistance is mainly attributed to the widespread use – and misuse – of antibiotics. When bacteria encounter antibiotics, those that carry rare mutations that make them more tolerant are able to reproduce, while susceptible ones die. Over time, descendants of the resistant variety will make up the entire population.

This does not just happen in patients, but in every environment where antibiotics are present: on farms, in fields, in waterways and around industrial sites that manufacture antibiotics. Often, the concentrations of antibiotics in these settings are too low to kill bacteria outright, but they are high enough to penalise those that are genetically more susceptible than others. Those that have even a small advantage reproduce faster, and populations evolve toward ever-higher resistance.

Bacteria are really small, so they can face huge variations in their environments moment by moment. Some of that variation consists of changes in the chemicals to which they are exposed. These may be a combination of food, secretions from neighbours, and toxins. The bacteria don’t consider whether the toxin is something that people call an antibiotic or a herbicide; they only care if it harms them. Bacteria can deal with these sub-lethal concentrations of stressful chemicals by ramping up their waste disposal systems.

Bacteria’s response to common weed-killers can also change their response to antibiotics

We decided to find out if other chemicals to which bacteria are exposed in the environment can affect their tolerance to antibiotics. We chose herbicides as examples of manufactured chemical products to which bacteria are exposed because they are used in almost every environment where people and bacteria co-exist. For example, in 2012, the most recent data available, the agricultural sector in the US alone used about 130,000 tonnes of glyphosate, the active ingredient in the weed-killer Roundup, 16,000 tonnes of 2,4-D, and 8000 tonnes of dicamba. These are also used by governments and municipalities in parks and along roads, and by gardeners and homeowners in their backyards. While these numbers are much lower than for agricultural use, they put many more people in potential contact with the herbicides.

We tested two potentially pathogenic bacterial species to find out whether exposure to weed-killers can make them more tolerant to antibiotics. Commercial formulations of common herbicides were used. They were based on the active ingredients dicamba, 2,4-D and glyphosate.

We saw that simultaneous exposure to an antibiotic and herbicide had two kinds of effects on bacteria compared with when they were only exposed to the antibiotic. In most cases, the bacteria were able to survive at higher concentrations of antibiotic. Sometimes, however, the bacteria became more susceptible and died even faster.

The effects we saw were caused by the activation of genes in what is called the “adaptive response”. The main contributors to the adaptive response are pumps that bacteria use to remove harmful substances. All bacteria have these genes, but they are only expressed when the proteins they code for are needed. Like the bilge pumps on a ship, they are only turned on when needed. Herbicides are just toxic enough to cause the bacteria to turn on these pump genes, and the pumps reduce the concentration of the herbicide inside the cell. This is sometimes enough to protect the bacterium.

How does this make the bacteria more resistant to more toxic chemicals such as antibiotics? Many of these pumps are not specific for one compound, but can expel a wide range of chemicals. Hence once they are induced by one chemical, the bacteria can better tolerate others too. This seems to be the case with herbicides and antibiotics.

We know that these pumps are involved in herbicide-induced effects because when we added a chemical inhibitor of these pumps, the protective effect caused by the herbicides disappeared. To narrow down which pumps were involved, we then used gene “knockout” strains that lacked individual pump genes. For several of these strains, both the antibiotics and the herbicides were much more toxic, and the protective effect disappeared. This told us that these genes were crucial for increased tolerance.

It is not just herbicides that cause changes to antibiotic tolerance

Herbicide formulations are complex mixtures of chemicals. In addition to the active ingredient that kills the plant, they contain substances that augment the effectiveness of the active ingredient by helping it to stick to the plant, even in rain, and be absorbed into the plant. These other ingredients may also ensure that the solution is homogenous, or bulk up the volume to usable amounts. Our initial experiments didn’t say which ingredient caused the effects on bacteria.

We tested the pure active ingredients and found that they caused the same pattern of responses we observed with the formulations: if the formulation caused an increase in anti­biotic tolerance, we usually saw the same with the active ingredient alone. Where we saw decreases in tolerance with the formulation, we also saw a decrease with the active ingredient.

The other substances in herbicides that we tested were Tween80 and carboxy-methyl cellulose (CMC). These also caused changes in the tolerance to antibiotics, but here the patterns we saw were sometimes different to what we observed with the commercial formulations. Because these other ingredients are not reported on the label of the commercial products, we cannot say whether the formulations we purchased contained Tween80 or CMC, or used other similar chemicals. Nevertheless, we can say for sure that both are used as emulsifiers in many other products as well, including processed foods or products like toothpaste or shampoo.

Where would this resistance matter? The answer depends on exposure and how much herbicide is used, but the recommended spray rate is more than enough to cause the effect.

If you are a farm animal, exposure can be through spray drift or eating herbicide-treated feed. Resistance might then occur in gut bacteria and be noticed when animals are also given antibiotics.

Interestingly, most of the antibiotic given to an animal is excreted, and excrement is often used as fertiliser. Hence crops and paddocks are fertilised with a combination of bacteria and antibiotics, and then may also be sprayed with herbicides, giving rise to resistant bacteria in soil. If you are a human, these anti­biotic-resistant bacteria may be on your lunch, having dropped off a fly that flew in from the paddock.

For the other chemicals, resistance-inducing concentrations were lower than is permitted in food. For people and domestic pets, ingesting processed food may be enough to cause the effect.

Contact exposure, though, is probably more important. Even petting your cat after it wandered through a neighbour’s freshly sprayed garden might be enough to introduce anti­biotic-resistant bacteria if you then touch your hand to your mouth.

We also found that the effects caused by different chemicals can be additive, which can decrease the amount of any individual chemical needed to induce changes in bacteria.

There are currently about eight million manufactured chemicals in commerce. Safety testing usually does not include effects on microbes, let alone sub-lethal ones. Hence, we have very little knowledge of how they or their breakdown products interact with bacteria, or how combinations of substances interact.

So what does this all mean for antibiotic resistance? It could mean that herbicides are accelerants when it comes to the evolution of antibiotic resistance. As David Bowie might say, if anti­biotic resistance is the fire, then using antibiotics and herbicides is like putting out fire with gasoline.