Dr Nilson C. Cruz

From

Technological Plasma Laboratory, São Paulo State University - BR

In residence at

Research Group in the Energetics of Ionized Media (GREMI) / CNRS, University of Orléans - FR

Host scientist

Dr Eric Robert

PROJECT

Non-antibiotics biocides produced by atmospheric plasmas with natural extracts

Antimicrobial resistance (AR), mostly caused by indiscriminate use of antibiotics, is an extremely worrying issue. In 2019, AR was associated to nearly 5 million deaths worldwide and it is estimated it could kill 10 million people per year in 2050. The reversion of such scenario, considered “terrible” by the World Health Organization in 2014, demands significant effort on multiple fronts, ranging from the establishment of proper antibiotics usage practices to the development of new antimicrobial agents. In this context, atmospheric pressure cold plasmas (APP) can be an immensely useful tool. With this technique, a myriad of reactive species is produced. Such species can interact with a material nearby or in contact with the plasma endowing it new chemical and physical characteristics. In previous studies we have observed that coatings produced by APP fed with natural extracts are highly efficient in inhibiting the adhesion and proliferation of several bacteria strains. Based on those promising results, it is proposed the application of APP jets fed with vapors of natural extracts, such as eugenol and carvacrol, to incorporate reactive species into water and physiological solutions, to render liquids with expressive antimicrobial properties. Additionally, the same experimental setup will be used to coat flexible materials with thin films derived from the extract to produce antimicrobial dressings. It will be evaluated the influence of treatment parameters on the antimicrobial potential of the treated media.
1. Introduction
The success of early antibiotic treatments was so remarkable that it led to the belief in the complete eradication of all infectious diseases. However, this sense of euphoria resulted in the widespread and indiscriminate use of antibiotics, leading to a significant problem that now threatens all therapeutic progress: bacterial resistance. Just two years after the commercialization of penicillin began, its discoverer was already warning about the risks of its excessive use. According to Fleming, bacteria would evolve, becoming resistant to antibiotics. As a result, drugs that were initially effective would become useless against microorganisms. Despite his warning, little was done to prevent such evolution, and in the 1940´s, the first reports of bacteria resistant to penicillin emerged. This led to the development of new bactericidal agents, such as vancomycin (in 1958), azithromycin (1980), ciprofloxacin, and more recently, in 2015, ceftazidime-avibactam. However, a recent report from the Center for Disease Control and Prevention identified bacteria resistant to all these antibiotics and several others not included in this list. This report, which links bacterial resistance to at least 38,000 deaths in the United States in 2017, lists microorganisms resistant even to carbapenem, which is reserved as the last resort in treating resistant pathogens. Worldwide, antimicrobial resistance killed 1.27 million people and was associated with about 5 million deaths in 2019.

The reversal of this scenario, classified by the World Health Organization in 2014 as terrible, requires significant effort on various fronts. In this sense, Cold Atmospheric Plasmas (CAP) should be considered as an enormously useful tool. In this type of discharge, the application of high electric fields can excite, ionize, and dissociate molecules of a gas or gas mixture, producing a myriad of highly reactive species containing oxygen (Reactive Oxygen Species – ROS) and nitrogen (Nitrogen Reactive Species – RNS) in the atmosphere surrounding the plasma. The importance of processes based on CAPs mainly results from the interaction of ROS and RNS with materials near the plasma. Since the action of plasmas essentially results in the introduction of highly reactive species into liquids, the investigation of the bactericidal action of plasma activated liquids has become practically compulsory. 
Given the fact that the modification of parameters used to generate the discharges strongly affects the type and proportion of the produced reactive species, it is practically impossible the microbes develop resistance against treatments with CAPs. Therefore, it becomes evident that plasma-activated liquids should be considered as important tool for the development of new biocides compounds. Owing to that, we propose to perform studies on the developing of non-antibiotic agents effective in reducing problems associated with bacterial resistance.
2. Main aspects to be investigated
The main objectives of this project are the acquisition of insights into how the characteristics of atmospheric pressure plasmas impact the chemical composition of various liquids and apply the acquired knowledge to develop technologies for adjusting these characteristics to enhance the bactericidal activity of treated liquids. Additionally, the same experimental setup will be used to coat flexible materials with thin films derived from the extracts to produce antimicrobial dressings. It will be evaluated the influence of treatment parameters on the antimicrobial potential of the treated media. 
3. Experimental procedure

This project will be developed in the laboratories of GREMI, Groupe de Recherches sur L´Energétique des Milieux Ionises at the University of Orléans. The choice of this group is due to its excellent infrastructure and the close affinity between the research conducted in both laboratories.
3.1 Sample treatment
The treatments will be applied to sterile vials containing 10 ml of the liquid to be modified. For this purpose, plasma jets produced by applying high-frequency pulses to two concentric cylindrical electrodes separated by a dielectric medium will be used. Discharges generated in argon, helium, nitrogen, and vapors of eugenol and carvacrol, either pure or mixed in different proportions, will be tested. The same experimental setup will be used to coat flexible membranes, such as hydrogels, for instance, with thin films produced by eugenol or carvacrol plasma polymerization. 
3.2 Sample Characterization
The characterization of the liquids will be conducted through high performance liquid chromatography and UV-Visible absorption spectroscopy employing specific reagent kits for each species to be determined. Morphology, chemical composition and structure of coated membranes will be evaluated by scanning electron microscopy, x-ray energy dispersive, x-ray photoelectron and Fourier Transform Infrared spectroscopies. Selected samples will undergo microbiological assays to evaluate the effect of incorporating species into the materials on the eradication of microorganisms.