Toward a better understanding of the microbial growth inhibition by electromagnetic fields

                                                                            PhD proposal – sept. 2017 to sept. 2020        

                                     Title : Toward a better understanding of the microbial growth inhibition by electromagnetic fields

Keywords :
Electromagnetism, microbial decontamination, growth inhibition mechanisms, numerical modeling

Labs :
– Institut de Microélectronique, Electromagnétisme et Photonique (IMEP-LAHC) http://imep-lahc.grenoble-inp.fr
Minatec – Grenoble  – 3, parvis Louis Néel, BP 257
38 016 GRENOBLE Cedex 1, FRANCE

– Institut des Géosciences de l’Environnement (IGE, UGA-CNRS-IRD-G-INP) http://www.ige-grenoble.fr
70 rue de la Physique, Bâtiment OSUG B , BP 53
38 041 GRENOBLE Cedex 09, FRANCE

PhD Director : XAVIER Pascal, pascal.xavier@univ-grenoble-alpes.fr, +33 (0)4.56.52.95.69,+33(0)4.76.82.53.65
PhD co-director : MARTINS Jean, jean.martins@univ-grenoble-alpes.fr, +33 (0)4.76.63.56.04

Funding : french research ministery doctoral grant (application must be made before march-april 2017)

Required skills and level of the applicant :
Master in biomedical or biophysical engineering. Some work experience in electronics are also desired. The proposed study is very large, from multiphysics modeling to experimental microbiological tests.

The transdisciplinary nature of this thesis provides skills in several domains as the design in analog and digital electronics instrumentation, testing in microbiology, multi-physics numerical finite element modeling. These skills will greatly be valued in a resume.
All necessary means for the progress of the work are already available within the two partner groups. The management team is composed of a university Professor and a CNRS Research Director, accompanied by two more Assistant Professors on instrumental aspects and modeling.

1.     Scientific context and objectives

In the battle against pathogenic microorganisms, in addition to the oldest curative process of pasteurization (heating) requiring large quantities of energy, current methods are mechanical actions (brushing) and the action of chemical products: acetic acid, hydrogen peroxide, chlorine dioxide… For example, the cheese industry is one of the largest users of chlorine. Unfortunately, some strains have become very resistant.
The use of physical means for the decontamination of water has only been explored for less than a century. Low intensity DC or AC current has been proven to be effective. This process was reported more than fifty years ago. Most articles in the literature focus on improving the effectiveness of antibiotics against microorganisms by applying weak currents, a phenomenon called “bioelectric effect” (Blenkinsopp 1992, Costerton 1994, Giladi 2008).
Several mechanisms have been proposed for this inhibition: electrolysis, production of toxic derivatives and free radicals linked to the electrodes, modification of the pH. In addition, the application of a high amplitude pulsed electric field has been used as a non-thermal effect for the inhibition of bacterial growth with the major disadvantage of the phenomenon of electroporation.
High-frequency electromagnetic fields (above MHz) but with small amplitudes (<1 V / cm) have also been reported as a means to improve the susceptibility of bacteria to antibiotics or to decrease their number in the absence of an antibiotic (Asami 2002, Bai 2006, Caubet 2004).
By exploiting this idea between 2011 and 2015, in the framework of the APELBIO project resulting from the ECO-INDUSTRY program of the French Ministry of Industry and carried out by the SME LEAS, in collaboration with SCHNEIDER ELECTRIC and two Grenoble laboratories involved in this project (IMEP-LAHC and IGE), we validated an innovative, non-polluting and energy-saving experimental concept for the prevention of microbial contamination in aqueous media . We noted that the optimal frequency for which this inhibition was maximal appeared to depend on the type of bacterium, which was confirmed by our numerical simulations using the COMSOL Multiphysics software with an original model (Xavier 2017). So we had the idea of using a white noise source (10kHz-10MHz) instead of a CW source. Our results, better than with a fixed frequency source, are in the state of the art and led to a patent in May 2015. Unfortunately, the fine mechanisms leading to the growth inhibition of bacterial cells could not be precisely identified. This is what we intend to do in the framework of this thesis project.

2. General issues

This doctoral work aims to contribute to a better understanding of the molecular mechanisms of the interactions between electromagnetic waves and biological cells in a context of microbial decontamination in liquids. The project is based on the recent work carried out within the framework of the APELBIO project cited above and seeks to identify the mechanisms of action of electromagnetic waves limiting the growth of micro-organisms in suspension (bacteria, yeasts and fungi, …). The different stages of doctoral work will therefore be:
1 / Design and realization of a compact instrument covering the 10 Hz – 50 MHz range for pilot experiments. This stand-alone instrument is based on the implementation of a DDS component in conjunction with a microcontroller. It will have the task of generating in a perfectly controlled manner the electromagnetic noise enabling the decontamination and, alternatively, of measuring the impedance detecting the decontaminating effect. A first prototype has already been developed recently and allowed us to carry out preliminary tests with the bacterium Escherichia coli.
The in situ detection of the decontamination efficiency requires a bio-impedance measurement of the solution containing the microorganisms. This last subject has, for many years, given rise to many patents and works: we know what toavoid to build a compact device, insensitive to the effects of electrodes
2 / Decontamination tests carried out following a wide range of physical conditions (amplitude and frequency of electromagnetic waves), chemical (variable geochemical environment, in terms of composition and strength ionic properties of the solution, which have an important effect on the surface properties of living cells, such as their zeta potential or their dispersed or agglomerated state which can potentially modulate electromagnetic effects) and biological (the type of bacterium studied could influence the electromagnetic effects already Observed on E. coli).
During the first year of the thesis, the doctoral student will establish a rigorous and reliable experimental plan which will allow to test all the factors initially identified as preponderant in the process of inhibition of the biological growth. From an experimental point of view, these tests will consist in treating cell cultures obtained under different conditions and culture media and in standardized conditions (same initial cell concentration, temperature, agitation, etc.). For each assay, cell growth and viability rates (flow cytometry, fluorescence microscopy, qPCR) and ATP synthesis (measured by bioluminescence and reflecting the cell physiological state) will be determined. Electromagnetic treatments (far below levels leading to thermal effects) will be carried out on selected bacterial models representative of different media and contexts (Escherichia coli, Pseudomonas sp, Salmonella anatum, Listeria sp., Bacillus subtilis, Listeria innocu … ). Tests with cell mixtures will also be conducted. In this case, molecular biology approaches will be implemented to monitor the effects of electromagnetic waves: genetic fingerprinting and cellular quantification by qPCR.
3 / Comprehension and numerical modeling under COMSOL Multiphysics of the mechanisms involved at the molecular and membrane level during the application of electromagnetic signals of low intensity. In our previous work, the model of the bacterium developed internally was simple. It is now necessary to refine this numerical model without, however, aiming at the complexity of the elaborated models used in synthetic biology, following two parallel paths, namely the modeling of microorganisms on the one hand and their environment on the other. The coupling and comparison of the results of modeling and microbiological follow-up of the decontamination tests should make it possible to identify the main mechanisms of action of the waves on the living cells.
As far as the environmental part is concerned, we wish to model realistically the behavior of the nutrient solutions in which the microorganisms are immersed, taking into account, in terms of electrical conduction and dielectric polarization, the various components of these solutions. Moreover, the modeling of the environment involves the fine study of the interface in the vicinity of the electrode.
The second major part of the proposed modeling work concerns the microorganism itself. We wish to pursue the approach that prevailed in our earlier work. Thus, the study previously carried out on E. coli has used a purely passive and dielectric shell model. This model made it possible to identify the frequency range leading to a maximum current absorbed by the microorganism, when an alternating voltage was applied to the medium loaded by the bacteria. Several improvements are needed today to refine the understanding of the phenomenon. First of all, it is necessary to take into account the presence of the charges (mostly protonic) involved in the bacterium, whether these are at rest or in motion: the bacterium becomes an active system. In the second place, it will be necessary to take into account the phenomena of mechanical vibrations, intervening in particular at the membrane level, since these also contribute to load shifts, the creation of electromagnetic fields or coupling with external fields.
To conclude on the modeling part, it should be noted that all these simulations are likely to lead to the development of an equivalent electrical network. This approach will make it possible, thanks to a systematic upstream study based on COMSOL Multiphysics, to treat general cases more simply by using free tools on the market (for example, SPICE software).

3. References

* IMEP-LAHC and IGE groups
Xavier P., D. Rauly, E. Chamberod and J.M.F. Martins. Theoretical evidence of maximum intracellular currents vs frequency in an Escherichia coli cell submitted to AC voltage. Bioelectromagnet. J. DOI:10.1002/bem.22033.
Archundia D., C. Duwig, L. Spadini, G. Uzu, S. Guédron, M.C. Morel, R. Cortez, Oswaldo Ramos, J. Chincheros, and J.M.F. Martins. How uncontrolled urban expansion increases the contamination of the Titicaca lake basin (El Alto – La Paz, Bolivia). Water, Air and Soil Pollution J. In press. 2017.
Navel A., L. Spadini, J.M.F. Martins, E. Vince and I. Lamy. Soil aggregates as a scale to investigate organic matter versus clay reactivities toward metals and protons. Accepted with revision. Eur. J. Soil Sci. 2017.
Archundia, D., C. Duwig, F. Lehembre, S. Chiron, M-C Morel, B. Prado, M. Bourdat-Deschamps, E. Vince, G. Flores Aviles and J.M.F. Martins. Antibiotic pollution in the Katari subcatchment of the Titicaca Lake: major transformation products and occurrence of resistance genes. Sci. Total Environ. 576 : (15) 671–682. 2017.
Ivankovic T., S. Rolland du Roscoat, C. Geindreau, P. Séchet, Z. Huang and J.M.F. Martins. Development and evaluation of an experimental and protocol for 3D visualization and characterization of bacterial biofilm’s structure in porous media using laboratory X-Ray Tomography. (GBIF-2016-0154). In press Biofouling J.
Simonin M., J.M.F. Martins, G. Uzu, E. Vince and A. Richaume. A combined study of TiO2 nano-particles transport and toxicity on microbial communities under acute and chronic exposures in soil columns. DOI: 10.1021/acs.est.6b02415. Environ. Sci. & Technol. 50: 10693–10699. 2016.
Simonin M., J. P. Guyonnet, J.M.F. Martins, M. Ginot and A. Richaume. Influence of soil properties on the toxicity of TiO2 nanoparticles on carbon mineralization and bacterial abundance. J. Haz. Mat. 283: 529-535. 2015.
D. Rauly, E. Chamberod, P. Xavier, J. M.F. Martins, J. Angelidis, H. Belbachir. First approach toward a modelling of the impedance spectroscopic behavior of microbial living cells, COMSOL Conference, Grenoble, 14-16 Octobre 2015
D. Rauly, E. Chamberod, P. Xavier, J. M.F. Martins, J. Angelidis, H. Belbachir, Stochastic Approach for EM Modelling of Suspended Bacterial Cells with Non-Uniform Geometry & Orientation Distribution, 36ème Progress In Electromagnetics Research Symposium (PIERS 2015), Prague (Rép Tchèque), 06-09/07/2015

* Others
Asami K. 2002. Characterization of biological cells by dielectric spectroscopy. Journal of Non-Crystalline Solids 305(1–3):268–277.
Blenkinsopp, A E Khoury, and J W Costerton. Electrical Enhancement of biocide efficay against Pseudomonas aeruginosa biofilms. Applied and Environmental Microbiology    Appl. Environ. Microbiol. November 1992 ; 58:11 3770-3773
Bai W, Zhao KZ, Asami K. 2006. Dielectric properties of E. coli cell as simulated by the three-shell spheroidal model. Biophysical Chemistry 122 :136–142.
Caubet R, Pedarros-Caubet F, Chu M, Freye E, de Belém Rodrigues M, Moreau JM, Ellison WJ. 2004. A radio frequency electric current enhances antibiotic efficacy against bacterial biofilms. Antimicrobial Agents and Chemotherapy 48(12):4662-4664.
Costerton JW, Ellis B, Lam K, Johnson F, Khoury AE. 1994. Mechanism of electrical enhancement of efficacy of antibiotics in killing biofilm bacteria. Antimicrobial Agents and Chemotherapy 38(12):2803-2809.
Giladi M, Porat Y, Blatt A, Wasserman Y, Kirson ED, Dekel E, Palti Y. 2008. Microbial growth inhibition by alternating electric fields. Antimicrobial Agents Chemotherapy 52(10):3517–3522.
Guiné V, Spadini L, Muris M., Sarret G., Delolme C., Gaudet JP, Martins JMF. 2006, Zinc Sorption to cell wall components of three gram-negative bacteria: a combined titration. Modelling and EXAFS study. Environ. Sci. Technol.  40 :1806-1813.

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