New research from DTU indicates that the outcome of a resistance measurement may depend on the conditions under which the bacterium is tested. Standard laboratory tests are carried out under fixed, uniform conditions, but if, for example, the test environment is altered, the very same bacterium may in some cases be either more or less susceptible to an antibiotic than the laboratory result indicates.
When doctors or veterinarians receive a laboratory report stating whether a bacterial sample is resistant to an antibiotic, the answer will typically be that the bacterium is susceptible (and can therefore be treated with antibiotics), or that it is not. That answer is correct for the standardised test conditions laboratories use, and it is this standardisation that allows results to be compared across laboratories.
However, standard conditions do not necessarily reflect all the environments bacteria encounter in real life. In the body (and across different hosts), factors such as pH level (how acidic or alkaline an environment is) and temperature can vary, and this may influence how effectively particular resistance genes function.
“We looked at two widespread resistance genes, and found that pH and temperature can greatly affect how well those genes function and therefore how susceptible the bacterium is to antibiotics. This could mean that a treatment may work in the body even though laboratory tests would suggest otherwise, and vice versa. Perhaps even more importantly, it may offer new clues as to how and why antimicrobial resistance develops and spreads in nature, in animals and between bacteria,” says Professor Frank Møller Aarestrup of the DTU National Food Institute.
Understanding how antimicrobial resistance develops and spreads is crucial, as antibiotic resistance has become an imminent threat to global public health.
Two resistance genes fluctuate in their susceptibility to antibiotics
In the study, the researchers investigated two widely prevalent resistance genes to determine how levels of resistance changed when pH and temperature were varied under controlled laboratory conditions. Among other measures, they quantified the amount of antibiotic required to kill the bacterium as pH was altered.
The researchers also examined the significance of temperatures comparable to the body temperatures of different hosts. Here, they observed an effect at temperatures corresponding to birds (around 42°C) compared with humans (around 37°C).
If a resistance gene functions better at 42°C than at 37°C (or vice versa), this may affect how readily bacteria carrying the gene survive and spread in birds, and thus the extent to which birds may act as hosts for bacteria with that type of resistance.
Monitoring birds in relation to antimicrobial resistance is important because birds can both acquire and disseminate resistant bacteria and resistance genes, while also reflecting the spread between the environment, agriculture and urban areas over long distances.
Better understanding
In the longer term, the research may therefore contribute to a better understanding of how resistance is expressed in different environments, and why resistance may develop and spread.
“The study can help us understand where and when particular resistance genes matter most, for example, in specific hosts, at particular temperatures, or in certain pH niches. So rather than only asking ‘is the resistance gene present here?’, we can increasingly also ask ‘under what conditions does it work best, and in which hosts are those conditions found? For instance, is it in birds or humans?’” says postdoc Mikkel Anbo of the DTU National Food Institute.
Far-reaching implications
The study was carried out in the laboratory on a limited set of material, and further research is needed before the researchers can say what else this new knowledge may be used for. The research raises a number of questions and possibilities, for example:
- First, it would be interesting to investigate whether the same applies to additional resistance genes, and not only the two tested in this study.
- In the longer term, the research could influence how we understand and interpret laboratory testing. The study shows that measured resistance levels can shift when pH or temperature changes. This does not mean standard tests are wrong, but it suggests that standard tests do not necessarily capture the full picture of how resistance genes behave under other conditions.
- The research also points to a possible future idea for urinary tract infections. In the experimental set-up, the resistance gene CTX-M-15 became markedly weaker at a more alkaline pH, and in some cases tipped from resistant to susceptible. This suggests it may be worth investigating whether, in urinary tract infections, it is possible to alter the environment in a way that helps combat resistant infections.
How the researchers did it
The researchers investigated whether acidity/alkalinity (pH) and temperature can change how well antibiotics work against bacteria carrying the resistance genes CTX-M-15 and CMY-2. The genes are found in several bacteria, including E. coli.
They used E. coli in the laboratory and altered the bacteria’s environment so that pH ranged from 5 to 9, corresponding to the variation in normal human intestinal pH. They also tested at different temperatures, including 37°C (as in the human body) and 42°C (as in birds). They then measured how much antibiotic was required to kill the bacteria.
What they found
- CTX-M-15 conferred the strongest resistance in acidic conditions and became weaker as the environment became more alkaline.
- CMY-2 performed better at more alkaline pH than CTX-M-15.
- At more alkaline pH, bacteria carrying CTX-M-15 could, in the experiment, shift from resistant to susceptible.
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Temperature also affected the results, which may be relevant when comparing different hosts and environments.
The scientific article Contrasting pH optima of β-lactamases CTX-M and CMY influence Escherichia coli fitness and resistance ecology has been published in the journal Applied and Environmental Microbiology. The research was funded by the Novo Nordisk Foundation and EU Horizon 2020.
Read more about The Research Group for Genomic Epidemiology.
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