Antibiotic resistance: a new mechanism observed in real time thanks to innovative microscopy techniques

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Fluorescence microscope image showing DNA transfer (yellow) through the conjugat
Fluorescence microscope image showing DNA transfer (yellow) through the conjugation pilus from donor bacteria (green) to recipient bacteria (red). Kelly Goldlust – Lesterlin LAB (MMSB, Lyon)

A better understanding of how bacteria acquire resistance to antibiotics is a key research issue in tackling the major public health problem of antibiotic resistance. The main mechanism by which these resistances are disseminated is called "DNA transfer by bacterial conjugation". Until now, this was thought to occur only between bacteria in direct contact with each other. In a new study, researchers from Inserm, CNRS and Université Claude-Bernard - Lyon 1, working in the Microbiologie moléculaire et biochimie structurale laboratory, have shed light on a new mode of resistance transfer between bacteria, demonstrating for the first time, using innovative microscopy techniques, that DNA transfer between physically distant cells is in fact possible. These results, and their many theoretical and clinical implications, are published in the journal PNAS.

Antibiotics considerably reduced mortality from infectious diseases during the 20th century, and thus represented a major advance in the field of medicine. However, for several years now, the problem of antibiotic resistance has been gaining ground. In France, some 5,500 deaths are linked to this phenomenon every year. Numerous research teams are now taking an interest in the subject, which has considerably increased our knowledge of the origins of antibiotic resistance.

These resistances can arise, for example, from a genetic mutation affecting the bacterium’s chromosome, or from the acquisition of foreign genetic material carrying one or more resistance genes from another bacterium.

In this second case, the transfer of DNA from the resistant "donor" bacterium to the "recipient" bacterium can take place via several mechanisms, the main one being known as "bacterial conjugation". This is at the heart of the research carried out by Christian Lesterlin, Inserm Research Director, and his team at the Molecular Microbiology and Structural Biochemistry Unit (CNRS/Université Claude-Bernard - Lyon 1).

For a long time, bacterial conjugation was described as a DNA transfer that could only take place when the donor bacterium was in direct physical contact with the recipient bacterium. Establishing this contact involves a "conjugation pilus", a small tubular appendage on the surface of the donor bacteria that enables attachment to a recipient bacterium.

"The pilus can be described as a kind of "molecular grabber" exposed on the surface of the donor bacterium and capable of extending to seek out and dock with a recipient bacterium. The pilus is then able to retract to establish membrane-to-membrane contact between the bacteria, prior to DNA transfer. However, 60 years ago, scientists proposed that this pilus could also act as a tunnel through which DNA could pass, allowing transfer to take place at a distance between two bacteria not in direct contact. But research aimed at obtaining direct proof of such a transfer was unsuccessful for a long time, leaving this hypothesis in abeyance" , explains Christian Lesterlin.

Until recently, there was no visualization technique enabling direct observation of DNA transfer between bacteria. With his colleagues, the Inserm geneticist therefore decided to use innovative fluorescence microscopy approaches, developed within his laboratory, to directly visualize conjugation between living cells. This type of approach had already borne fruit the first time in 2019, when the team observed live the acquisition of antibiotic resistance by an E. Coli bacteria [1] .

In this new study, researchers have developed a technique for visualizing, in real time and for the first time, DNA transfer across the extended pilus, which establishes contact between two physically distant bacteria.

" Our microscopic observations show unequivocally that the pilus has a dual function. It establishes direct contact between two cells, but it can also act as a conduit for DNA during transfer between physically distant cells. These results help to update our knowledge of resistance transfer by bacterial conjugation, showing that, in some cases, bacteria do not need to be in direct contact for DNA to be transferred and resistance dissemination to occur" , emphasizes Christian Lesterlin.

This work is helping us to better understand the mechanisms by which antibiotic resistance spreads. Indeed, knowing that two physically distant bacteria can exchange DNA means that resistance transfers can take place in different environments where direct contact between bacteria is made more difficult by the complexity or viscosity of the environment, such as in the intestine.

Finally, by shedding light on a hitherto poorly characterized mode of DNA transfer, this work could in the longer term pave the way for the development of therapeutic tools aimed at targeting and inhibiting these mechanisms of transmission of antibiotic resistance between bacteria.

[1] S. Nolivos et al, Science, May 24, 2019; doi: 10.1126/science.aav6390