Estudio de la actividad del plasma frío a presión atmosférica sobre el biofilm y células planctónicas de Staphylococcus aureus, Pseudomonas aeruginosa y Candida albicans

The purpose of this thesis is to study the activity of cold plasma or Atmospheric No thermal plasma (ANTP) on biofilm and the planktonic forms of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. Plasma constitutes the fourth state of aggregation of matter, after solids, liquids and gases. It is produced by completely or partially ionizing a gas, being able to use any source of energy for its generation (electrical, thermal, optical or ionizing electromagnetic radiation). When free electrical charges (electrons and positive ions) occur, plasma is a good conductor of electricity. The plasma state is reached when a large part of the particles that make up the gas reach enough energy to allow its ionization to release a high electron density. The produced electrons and their subsequent collisions with the atoms and molecules of the gas generate the reactive species that will interact with the environment. Plasma can be classified according to the relative energy levels of electrons and heavy particles (ions and neutrals) in thermal plasma and non-thermal plasma also called non-equilibrium plasma or cold plasma. In thermal plasma, high temperature implies that all chemical species (Te≈Ti≈Tg ) are in a state of thermodynamic equilibrium. In non-thermal plasma, the cooling of ions and molecules without charge, is more effective than the transfer of energy of electrons in collisions, the thermal equilibrium is not achieved and the gas remains at low temperature (Te>>Ti≈Tg ) so is known as cold plasma. Cold plasmas are capable of destroying, at room temperature, microorganisms with minimal or no heat damage. Cold plasmas can be generated in vacuum chambers, called low pressure plasmas, or at atmospheric pressure called atmospheric pressure plasmas. In those generated at low gas pressures, the frequency of collision of the electrons is low, so that the energies of the electrons will remain high compared to the energies of the heavy particles (ions and neutrals) representing a state of non-equilibrium plasma. At atmospheric pressures the frequency of collisions of electrons is higher, so the difference between Te and the temperature of the heavy particles will decrease and therefore, the state of the plasma will be closer to the state of thermal equilibrium. This condition can be avoided by providing a low power supply density or a pulsed power, which leads to the formation of cold or non-equilibrium plasma. The need to biologically decontaminate sensitive material or to sterilize material in operating rooms has made for years that various decontamination techniques are studied. There are various techniques used today such as dry heat, wet heat, ultraviolet radiation, hydrogen peroxide or liquids with various chemical compositions, but none can be applied to living tissues or especially sensitive materials. The use of cold plasma at atmospheric pressure emerges as a relatively new technique that allows decontamination at room temperature and atmospheric pressure, living tissues or especially sensitive materials. It seems to be a good candidate for microbial inactivation in biofilms or the destruction of the structure in an ecological and cost-effective manner. The oxidative stress produced by plasma chemistry reagents is responsible for the effects that occur. Since my professional activity is dedicated to the treatment of prosthetic joint infection and chronic osteomyelitis in adults, where biofilm is the cause of chronicity and difficulty in curing these diseases, the possible application of cold plasma to decontaminate or sterilize living tissues and prosthetic material motivated my interest in the subject and the development of this doctoral thesis. For the development of the of the experimental work we used a device called the Atmospheric Pressure Plasma Chamber - Non-Thermal Plasma (APPC-NTP) characterized by using air as a gas, working at room temperature and atmospheric pressure for plasma production, making it a possible candidate to be used economically in a multitude of situations, including in the field of septic surgery of the musculoskeletal system. The calibration of the equipment was already carried out by PhD. Guillermo Baeza Oliete in his thesis “Contribution to the generation of cold plasma using SMD and JET electrodes”. In order to demonstrate the antimicrobial capacity of the plasma generation chamber on biofilms from S. aureus, P. aeruginosa and C. albicans formed on commercially pure titanium (grade 3) and polytetrafluoroethylene (PTFE / Teflon) discs and on their respective cells planktonic, we developed four experimental designs. In the first two experimental designs, we evaluated the activity of cold plasma on the biofilm of S. aureus, P. aeruginosa and C. albicans formed on commercially pure titanium disks and PTFE and on their respective plankton cells. In the third experimental design, the ability of cold plasma to act on the culture medium and prevent the subsequent growth of microorganisms is analyzed. In the last experimental design, the viability of the biofilm of S. aureus, P. aeruginosa and C. albicans was evaluated qualitatively using laser confocal microscopy after exposure to cold plasma. The average lethality produced on the biofilm of S. aureus, P. aeruginosa and C. albicans, formed during 48 hours on titanium discs, after 60 minutes of exposure to cold plasma was 99.7%, 99.9% and 99.6%, respectively. In the case of PTFE discs, it was 99.1%, 95.9% and 94.4%, respectively. The mean lethality of the planktonic cells of S. aureus, P. aeruginosa and C. albicans after exposure of cold plasma for 90 minutes was 99.87%. Exposure of the BHI culture medium to cold plasma for 90 minutes generates a plasma activated culture medium (PAM) that prevents subsequent growth of S. aureus, P. aeruginosa and C. albicans. We verified, by confocal microscopy of laser exploration, that after exposure to cold plasma for 60 minutes of glass disks on which a 48-hour biofilm of S. aureus, P. aeruginosa and C. albicans had previously formed, produced a complete destruction of the biofilm.