The extraordinary powers of bacteria visualized in real time

The global spread of antibiotic resistance is a major public health problem and a priority for international microbiology research. In an article to be published in the journal Science, a team led by Christian Lesterlin, Inserm researcher CNRS / University Claude Bernard Lyon 1, filmed the process of acquiring antibiotic resistance in real time, discovering an unexpected key player in its maintenance and its spread within bacterial populations.

This spread of antibiotic resistance is largely due to the ability of bacteria to exchange genetic material through a process called bacterial conjugation. Systematic sequencing of pathogenic or environmental strains has identified a wide variety of genetic elements that can be conjugated and resistant to most, if not all, clbades of antibiotics currently used in clinical settings. . However, the process of transferring genetic material from one bacterium to another in vivo, the time required to acquire this resistance once the new genetic material is received and the effect of the antibiotic molecules on that resistance have not been elucidated.

Real time visualization

The researchers chose to study the acquisition of Escherichia coli resistance to tetracycline, a commonly used antibiotic, by placing a tetracycline-sensitive bacterium in the presence of a resistant bacterium. Previous studies have shown that such resistance involves the ability of the bacteria to expel the antibiotic before it can exert its destructive effect by means of efflux pumps found on its membrane. . These specific efflux pumps are able to eject the antimicrobial molecules from the bacteria, giving them a certain level of resistance.

In this experiment, the transmission of DNA from a specific "efflux pump" – the TetA pump – was observed between a resistant bacterium and a sensitive bacterium with the help of a fluorescent marking. Through live cell microscopy, the researchers simply had to follow the progress of the fluorescence to see how the DNA of the "pump" migrated from one bacterium to another and how it was expressed in the recipient bacteria.

The researchers found that within one to two hours, the single-stranded DNA fragment of the efflux pump was transformed into double-stranded DNA and translated into functional protein, thereby conferring resistance to the tetracycline of the recipient bacterium.

The transfer of DNA from the donor bacteria (green) to the recipient bacteria (red) is revealed by the appearance of red locating foci. The rapid expression of newly acquired genes is revealed by green fluorescence production in receptor bacteria.

How is resistance organized in the presence of an antibiotic?

Scientists are well aware of the mode of action of tetracycline: it kills bacteria by binding to their translation mechanism, thus blocking any possibility of protein production. Following this reasoning, one would expect that by adding the antibiotic to the previous culture medium, the TetA efflux pump would not be produced and the bacteria would die. However, the researchers observed that, paradoxically, bacteria were able to survive and effectively develop resistance, suggesting the involvement of another factor essential to the process of acquiring resistance.

Scientists have discovered that this phenomenon can be explained by the existence of another efflux pump, present in almost all bacteria: AcrAB-TolC. Although this general-purpose pump is less effective than TetA, it is still able to expel a small amount of antibiotic from the cell, which means that the bacteria can maintain minimal protein synthesis activity. Therefore, if the bacterium is lucky enough to have received a resistance gene by conjugation, the TetA pump is produced and the bacterium becomes resistant in a sustainable manner.

This study opens new avenues in the search for similar mechanisms in bacteria other than E. coliand for different antibiotics. "One could even consider a therapy combining an antibiotic and a molecule capable of inhibiting this generalist pump.It is still too early to consider the therapeutic application of such an inhibitor, many studies are currently underway in this area antibiotic resistance and prevent its spread to different bacterial species ". concludes Lesterlin.