Visualizing how an adjuvant boosts the effectiveness of antibiotics against a resistant bacterium

A collaborative study between Aix-Marseille Université, SOLEIL, ESRF and ALBA synchrotrons, recently published in npj Antimicrobials and Resistance, provides new insight into the mechanism of action of NV716, an antibiotic adjuvant capable of restoring the activity of certain antibiotics against the multidrug-resistant bacterium Pseudomonas aeruginosa.

Gram-negative bacteria such as P. aeruginosa exhibit strong intrinsic resistance due to the low permeability of their outer membrane and the efficiency of their antibiotic efflux systems. Certain adjuvants, such as NV716, enhance the effectiveness of antibiotics against these resistant bacteria. But how do they work?

To understand the mechanisms involved, several complementary synchrotron imaging approaches were combined. At the DISCO beamline of Synchrotron SOLEIL, deep ultraviolet (DUV) microspectrofluorimetry experiments enabled real-time monitoring of antibiotic accumulation (DUV-fluorescent antibiotics) in individual bacteria using a dedicated microfluidic device developed for the study (Fig. 1). This device greatly improves the monitoring of antibiotic accumulation in bacteria using UV microscopy, by allowing to record the initial stages of accumulation and to overcome the issue of bacterial mobility before and during the uptake process.

DISCO-POLARIS-adjuvant-fig1 — *Figure 1: Real-time monitoring of antibiotic (doxycycline) accumulation in* Pseudomonas aeruginosa by deep ultraviolet (DUV) microspectrofluorimetry at the DISCO beamline of SOLEIL. Experiments were performed using a microfluidic device developed to track antibiotic entry into individual bacteria exposed to the NV716 adjuvant.

Cryo-soft X-ray tomography analyses performed at the MISTRAL beamline of the ALBA Synchrotron then revealed membrane alterations in bacteria exposed to the adjuvant, associated with increased production of outer membrane vesicles (OMVs) (Fig. 2).

DISCO-POLARIS_adjuvant-fig2 — *Figure 2: Visualization by soft X-ray cryotomography (cryo-SXT, MISTRAL beamline – ALBA) of membrane alterations induced by NV716 in* Pseudomonas aeruginosa. Tomographic reconstructions show untreated control bacteria (left) and bacteria exposed to NV716 for 30 minutes (right). The treatment induces increased production of outer membrane vesicles (OMVs), visible at the bacterial surface (arrows). Scale bar: 1 µm.

These observations were complemented by cryo-electron microscopy experiments carried out at SOLEIL (POLARIS platform), enabling visualization of the ultrastructure of vesicles produced in the presence of NV716 (Fig. 3).

DISCO-POLARIS-adjuvant_fig3 — Figure 3: Cryo-electron microscopy imaging (cryo-EM, POLARIS platform – SOLEIL) of bacterial membrane vesicles isolated after NV716 treatment. The vesicles display strong heterogeneity in size and morphology, suggesting different forms of outer membrane remodeling in response to the membrane stress induced by NV716. Scale bar: 50 nm.

Finally, X-ray fluorescence nano-imaging experiments conducted at the ID16A beamline of the ESRF enabled localization of a copper-containing derivative of the adjuvant (Cu-NV716) at the periphery of the bacteria, confirming its association with bacterial membranes (Fig. 4).

DISCO-POLARIS_fig4 — Figure 4: X-ray fluorescence nano-imaging performed at the ID16A beamline of the ESRF showing the peripheral localization of a copper derivative of the adjuvant at the surface of Pseudomonas aeruginosa. Panels A and C show phosphorus distribution, used as a marker of the bacterial cell, whereas panels B and D show the distribution of copper associated with the Cu-NV716 derivative. Panels A and B correspond to untreated control bacteria; panels C and D correspond to bacteria exposed to Cu-NV716.

These results show that NV716 acts primarily by disrupting the organization of the outer membrane, thereby promoting intracellular accumulation of antibiotics such as doxycycline.

Beyond elucidating the mode of action of NV716, this work opens new perspectives for exploring the mechanisms of OMV biogenesis and for developing strategies aimed at modulating membrane permeability in multidrug-resistant bacteria.