Identification and characterization of chemical/natural adjuvants to decrease the overuse of antibiotics in veterinary medicine

L’émergence de bactéries multirésistantes aux antibiotiques conduit la médecine humaine et vétérinaire à des impasses thérapeutiques. L’usage abusif des ATBs accélère et accentue fortement le processus d’antibiorésistance. Le but de mon travail de thèse était d’apporter une solution au problème de surconsommation d’ATBs en médecine vétérinaire, particulièrement dans les élevages, tout en garantissant la santé animale. Nous avons développé une approche de thérapie combinatoire basée sur l’association d’ATBs déjà existants avec des molécules adjuvantes. Nous avons ainsi évalué l’activité de différents dérivés polyaminés d’origine naturelle sur des pathogènes dits « ESKAPE » qui présentent une relevance clinique majeure en médecine humaine. Ces différentes études ont permis de développer des protocoles de criblage, mais également des méthodes permettant d’élucider leur impact sur la physiologie bactérienne.Nous avons testé l’activité de ces composés en combinaison avec le florfénicol, ATB couramment utilisé pour traiter les infections respiratoires chez le porc. Après avoir identifié un adjuvant au florfénicol, nous avons pu déterminer son mode d’action par différentes méthodes de fluorescence et de bioluminescence. Nous avons pu démontrer que le composé permet de potentialiser l’activité de l’ATB par une action membranotrope et par une inhibition de l'efflux, augmentant ainsi la concentration intracellulaire en ATB, mais également par une inhibition de la force proton motrice (FPM). La motricité flagellaire des bactéries, est un facteur de virulence fonctionnant avec la FPM nous avons pu mettre en évidence une inhibition de la mobilité par le composé.The constant increase of multidrug resistant bacteria is leaving clinicians and veterinarians with very limited options to treat bacterial infections. The main goal of my work was find a chemical solution to reduce the use of antibiotics in veterinary medicine, especially in swines, without affecting the health of the livestock. To achieve this goal, we have developed a drug combination approach based on the association of antibiotics with chemosensitizers, herein called adjuvants, some of which were polyamines derivatives from natural sources. To provide the proof of concept, combination of several derivatives from different chemical sources (marine sponges and plants) have been tested ex vivo on « ESKAPE » pathogens, which are among the most urgent bacterial threats. Results from these studies allowed us to develop procedures for screening antibacterial activities and methodologies for understanding the impact of the selected adjuvants on bacterial physiology.Florfenicol is a widely used antibiotic to treat respiratory infections in swine. Therefore, derivatives were further assessed in combination with florfenicol, and florfenicol adjuvants were identified. The mode of action of one chosen adjuvant on bacterial membranes was further investigated by using fluorescence and bioluminescence methods. Data showed that this molecule was able to potentiate the antibiotic activity by increasing its intracellular concentration (membranotrope activity and inhibition of efflux) but also causes inner membrane depolarization. Flagellar motility represents an important virulence factor which use PMF, and we showed a diminution of the motility's halo with our compound

Polyamine derivatives: a revival of an old neglected scaffold to fight resistant Gram-negative bacteria?

International audienceEmergence of multidrug resistant pathogens was responsible for microbial infections and inefficacy of numerous antimicrobial therapies has induced a need for the research of new classes of antibiotics. In this review, we will focus our interest towards the biological properties of polyamino antimicrobial agents

Polyamino-isoprenyl derivatives as antibiotic adjuvants and motility inhibitors for 1 Bordetella bronchiseptica porcine pulmonary infection treatment 2 3

International audienceThe spreading of multidrug-resistant bacteria and the lack of novel antibiotic molecules leave clinicians and veterinarians with very limited options to treat bacterial infections, especially those caused by Gram-negative pathogens. To reduce the selection of antibiotic resistance mechanisms and their transfer to human pathogens, veterinary pharmaceutical companies have dramatically decreased the number of antibiotics used. Among all the investigated alternate solutions, chemosensitizers, which decrease the amount of the used drugs, appear to be one of the most promising strategies. In this study, we reported that polyamino-isoprenyl derivatives can potentiate florfenicol activity against veterinary sensitive reference strains as well as clinical isolates. These molecules induce inner membrane depolarization and subsequently inhibit efflux pumps by collapsing the proton-motive force (PMF). Considering that Bordetella bronchiseptica rotor flagellum is highly PMF dependent and that flagellar motility represents an important factor involved in colonization, we monitored the swimming and swarming motilities of bacteria and showed a strong inhibition in the presence of the lead selected compound. Taken together, our results suggest that this class of molecules are able to increase treatment efficacy and decrease drug consumption

Motuporamine Derivatives As Antimicrobial Agents And Antibiotic Enhancers Against Resistant Gram-Negative Bacteria

Dihydromotuporamine C and its derivatives were evaluated for their in vitro antimicrobial activities and antibiotic enhancement properties against Gram-negative bacteria and clinical isolates. The mechanism of action of one of these derivatives, MOTU-N44, was investigated against Enterobacter aerogenes by using fluorescent dyes to evaluate outer-membrane depolarization and permeabilization. Its efficiency correlated with inhibition of dye transport, thus suggesting that these molecules inhibit drug transporters by de-energization of the efflux pump rather than by direct interaction of the molecule with the pump. This suggests that depowering the efflux pump provides another strategy to address antibiotic resistance

Polyamino-Isoprenic Derivatives Block Intrinsic Resistance of P. aeruginosa to Doxycycline and Chloramphenicol In Vitro

International audienceMultidrug resistant bacteria have been a worldwide concern for decades. Though new molecules that effectively target Gram-positive bacteria are currently appearing on the market, a gap remains in the treatment of infections caused by Gram-negative bacteria. Therefore, new strategies must be developed against these pathogens. The aim of this study was to select an antibiotic for which a bacterium is naturally resistant and to use an escort molecule to restore susceptibility, similarly to the model of β-lactam/ β-lactamase inhibitors. High-content screening was performed on the reference strain PA01, allowing the selection of four polyamino-isoprenic compounds that acted synergistically with doxycycline. They were assayed against clinical isolates and Multi-Drug-Resistant strains. One of these compounds was able to decrease the MIC of doxycycline on the reference strain, efflux pump overpro-ducers and clinical isolates of P. aeruginosa, to the susceptibility level. Similar results were obtained using chloramphenicol as the antibiotic. Membrane permeation assays and real-time efflux experiments were used to characterize the mechanism of doxycycline potentiation. The results showed that the selected compound strongly decreases the efficiency of glucose triggered efflux associated with a slight destabilization of the outer membrane. According to these data, targeting natural resistance may become an interesting way to combat MDR pathogens and could represent an alternative to already devised strategies

2,4‐Disubstituted 5‐Nitroimidazoles Potent against Clostridium difficile

International audienc

Claramines: A New Class Of Broad-Spectrum Antimicrobial Agents With Bimodal Activity

International audienceThe emergence of multidrug resistant bacteria/pathogens has highlighted the need for the development of new antibiotics. In this manuscript, we report herein our results dealing with the broad spectrum of antibacterial activity against both sensitive and resistant Gram-negative and Gram-positive bacterial strains of an easily prepared water soluble polyaminosterol compound namely claramine A1. We will also demonstrate its peculiar mechanism of action (different from all the well-known classes of antibiotics) towards Gram-negative and Gram-positive bacteria. Finally, due to its low cytotoxicity, this class of molecules could constitute an effective response to combat the emergence of multidrug resistant bacteria and nosocomial diseases

Membrane permeation assays.

<p>The effect on the PAO1 strain of compound <b>3</b> (black-filled and black-hatched bars) and PMBN (grey-filled and grey-hatched bars) were compared at concentrations ranging from 15.6 μM to 125 μM, as indicated. The relative effect corresponds either to efflux inhibition (filled-bars) or to outer-membrane permeation (hatched-bars). Error bars represent standard deviations of 2 independent experiments. * indicates statistically significant (p ≤ 0.05) differences of relative effect (efflux inhibition/outer membrane permeation) for compound <b>3</b> treatment compared to the respective PMBN treatment. Brackets indicate statistically comparable permeation effects.</p

Inhibition of the efflux of the 1,2’ dinaphthylamine dye by the compound 3.

<p>Efflux was triggered after 200 s by the addition of 50 mM glucose (arrow). The intensity of fluorescence emission for 1,2’ dinaphthylamine is given in relative fluorescence units (RFU). (a) Concentration-dependent inhibition of 1,2’ dinaphthylamine efflux by compound <b>3</b> after glucose addition (arrow). Compound concentration (μM) is indicated in front of each green curve; as a control, an experiment without addition of compound and glucose (-glc, red curve) was also included; (b) percent of efflux inhibition obtained for each compound and PAßN as indicated. Error bars represent standard deviations of 2 independent experiments. * indicates statistically significant difference in values (p < 0.05) compared to PAßN.</p

Structure of polyamino-isoprenic derivatives 1–4 identified in this study.

<p>LogD have been determined by using chemical simulation software Marvin Sketch 5.11.3.</p