On the DISCO beamline: how to resist resistant bacteria…

The intensive and sometimes injudicious use of antibacterial agents- antibiotics, disinfectants, etc, leads to the selection of bacterial populations resistant to the molecules used, even without having had direct contact with these molecules. In fact, so-called “naive” bacteria possessing no mechanism of protection against these different products no longer exist. Until recently, scientists used the term Multi Drug Resistance, or MDR, but now they talk of XDR; totally multi-resistant bacteria, capable of resisting all antibiotic groups and even molecules only just being discovered by man and not yet in use…  

Without wanting to be alarmist, it is simply realistic to consider the importance, and even the urgency of this problem, which Jean-Marie Pagès has been working on since 2000, in collaboration with the “Transporteurs Membranaires, Chimiorésistance et Drug-Design” laboratory in Marseille.

An antibiotic has to be present in bacteria in sufficient concentrations to be effective. As it happens, the first line of defense set up by bacteria consists of limiting the concentration of the active principle, by reducing membrane permeability and/or expelling the molecule after it has penetrated. Only small quantities of administered antibiotic are present in bacteria, i.e. a concentration that is not immediately effective. This therefore provides time for bacterial mechanisms to be triggered that produce enzymes capable of destroying antibiotics (second line of defense). Finally, due to the intervention of this membrane barrier, bacteria can make use of a third weapon: modifying the target molecule of the antibiotic through mutation of the DNA that carries its genetic information, or hide the binding site for the antibiotic on this molecule through the production of another masking molecule. As the target is no longer recognized, the antibiotic becomes inactive. This protective chain reaction can be induced not only by antibiotics but also by certain molecules present in the products cited earlier that are supposed to protect us from bacteria.

This impressive adaptability stems from the natural evolution of procaryotes and their millions of years of confrontation with environmental aggressions. Man, on the other hand, has only had 60 years of experience in the use of antibiotics.

J-M Pagès and his group have focused part of their research on the diffusion of antibiotics across the bacterial membrane, as this is the first stage in antibiotic resistance. However, although we know that only a small proportion of the antibiotic penetrates effectively into resistant bacteria, we still cannot measure this intra-bacterial concentration in a precise and reliable way.

The standard methods used are based on the use of radioactive-labeled antibiotics, which are difficult and costly to buy and besides, their manipulation has the disadvantages associated with the fact that these are radioactive products. In fact, the intrabacterial antibiotic concentration is calculated by measuring the radioactivity in these bacteria, then comparing it with the total quantity of bacteria in the culture (including those that have not “ingested” the antibiotic). This total quantity is evaluated based on a control culture prepared in parallel and under the same conditions, but without antibiotics. Using this method, the number of bacteria incubated with the antibiotic in suspension is obtained, thus providing the quantity of antibiotic in relation to the number of bacteria. In addition, by measuring the proteins in the sample (by colorimetric assay), the quantity of antibiotic in relation to the quantity of bacterial protein can be calculated. However the experimental work associated with these techniques is a source of errors, leading to problems in precision and reproducibility of results.

In addition, if we would like to know in which compartment in bacteria the antibiotic is located, these bacteria have to be broken apart. This stage could give false results as the molecules released are often disseminated to compartments other than those where they were initially found.

It is for this reason that the Marseille group is perfecting a new technique to measure the concentration and localization of antibiotics in bacteria in collaboration with Matthieu Réfrégiers’ group on the DISCO beamline.

Rather than add a marker on the antibiotic that can be tracked, the idea is to detect it under its native non-modified form – which would eliminate all possible artifacts due to the marker. The solution is therefore to measure a property that the antibiotic under study possesses naturally, as it happens, the fluorescence that it emits after excitation with a UV beam with a sufficiently long wavelength. This is where the DISCO beamline comes into play.

The experiment then becomes simpler and more reliable than the one previously described: once the wavelength required to excite the antibiotic has been determined, the measurement is carried out on a sample of the bacterial culture. Illuminated by the UV beam, the emission spectrum obtained is composed of peaks corresponding to the fluorescent molecules found in bacteria: the associated antibiotic, but also certain bacterial components (notably proteins containing aromatic amino acids). With one analysis it is therefore possible to measure, based on to the intensity of the different peaks, the quantity of intra-bacterial antibiotic, as well as a sum representing the total quantity of bacteria. The great advantage of this technique is that there is no longer any need for “control cultures” as all measurements are carried out on the same sample!

JM Pagès, his collaborators and the DISCO team showed that this intrinsic fluorescence measurement was a very good inhouse standard against which to compare later antibiotic fluorescence peaks under different experimental conditions. They have tested bacteria susceptible to antibiotics, bacteria that are resistant to antibiotics due to modifications to membrane transporters involved in antibiotic penetration or expulsion and finally resistant bacteria to which a component inhibiting resistance has been added, in order to evaluate its efficacy.

As it is, this experimental protocol, in the process of validation, already presents advantages when compared to standard methods. This is not all: by combining microscopy with fluorescence spectroscopy and because of the intensity and size of the DISCO beamline, it should be possible not only to assay the antibiotic in the bacterium, but also to localize it precisely to a particular compartment of the bacterium: membrane, cytoplasm, etc.

The aim of this research, in the long term, is to obtain a molecular definition, in situ, of the antibiotic resistance mechanism. New biophysical tools that, when added to the studies carried out by chemists, geneticists and biologists, will help to strengthen the arsenal of techniques aimed at decrypting the active resistance mechanisms in bacterial infections.