FR3036708A1 - METHOD OF INHIBITING THE DEVELOPMENT OF MICROORGANISMS - Google Patents

Abstract

The invention relates to a device and a method for inhibiting the development of microorganisms, such as bacteria, in a medium, and in particular a liquid medium. More specifically, the method comprises applying a bias voltage to the liquid medium, the frequency of said voltage varying, in a time interval of less than 1 hour, between a minimum frequency and a maximum frequency, the maximum frequency being at least twice the minimum frequency.

Description

TECHNICAL FIELD The invention relates to the inhibition of the development of microorganisms, for example bacteria, in a medium, and in particular a liquid medium. The treated liquid may in particular be waste water from a domestic, industrial or hospital installation, or any other liquid, including liquids for agri-food use.

Description of the Prior Art In order to limit the use of chemicals, the use of physical methods to limit the development of pathogenic microorganisms, such as bacteria, has been studied for several decades. Indeed, it turns out that certain methods have an inhibitory effect on the development of these microorganisms. Among the investigated methods are the application of ultrasonic waves, light, a temperature gradient or a magnetic field. The application of weak, continuous or low frequency electric currents has also been shown to be effective. For example, in Costerton, "Mechanism of Electrical Enhancement of Efficacy of Antibiotics in Killing Biofilm Bacteria," Antimicrobial Agents and Chemotherapy, Dec. 1994, it has been shown that the application of direct current to the surface of an electrode metal used to remove a bacterial biofilm formed on this surface. Moreover, this publication demonstrates a remarkable efficiency in the combined application of a continuous electric current and an antibiotic. This synergistic effect is referred to as the "bioelectric effect".

But the application of direct currents can release ions in the liquid medium, by electrolysis, the latter being toxic to healthy cells. Recently, the combination of an alternating electric field and an antibiotic to inhibit the growth of bacteria has been studied. For example, patent application 3036708 2 US2012 / 0184895 describes the application of an alternating electric field of low amplitude (a few V / cm) and an antibiotic on bacteria of Staphylococcus aureus and Pseudomonas aeruginosa type. In this document, bacteria immersed in a culture medium are placed between two electrodes between which an alternating voltage is applied whose frequency is successively set at 0.1 MHz, 1 MHz, 5 MHz, 10 MHz, 20 MHz, 30 MHz. and 50 MHz. At each frequency, a test lasting several hours is carried out, and the evolution of the bacterial population is monitored by optical density measurements, using a spectrophotometer, on samples taken.

The paper concludes that the frequency of 10 MHz allows maximum inhibition of bacterial growth for both bacteria studied. This effect is attributed to the action of electrostatic forces, in particular dielectrophoretic forces acting on the cell membrane during cell division. These forces seem in particular to act on the constriction forming between the two daughter cells, during the telophase, and to induce a rupture of the cell membrane. When the dividing cell is oriented parallel to the field lines, the intensity of the forces is maximum. In parallel, modelizations of these forces have been realized, highlighting a maximum intensity at a frequency of 2 MHz for Pseudomonas aeruginosa, and at a frequency of 7 MHz for Staphylococcus aureus.

Furthermore, this patent application indicates a decrease in the minimum inhibitory concentration of an antibiotic, in this case chloramphenicol, placed in the culture medium, in the presence of an electric field of frequency equal to 10 MHz. It appears from this document that the application of an alternating electric field has undeniable effects on the limitation of bacterial proliferation, but that there is an optimal frequency for each type of bacterium. Thus, each type of microorganism seems to have its own "frequency sensitivity", which must be determined by tests similar to those described above. Indeed, nothing says that an optimal frequency for one microorganism will be optimal for another microorganism. Moreover, it has not been established that an optimal frequency with respect to a microorganism in a given medium will also be optimal, with respect to the same microorganism, in another medium. Moreover, in industrial plants, one can encounter different types of microorganisms, and the purification techniques must target all the microorganisms likely to be encountered. The method recommended in the document described above, if it seems effective for Pseudomonas aeruginosa and Staphylococcus aureus, will not necessarily be effective for other species. A method for treating a medium, in particular a liquid medium, is therefore sought, aimed at limiting bacterial growth, or even at reducing the concentration of microorganisms, without requiring their prior identification, and by limiting the use of chemical agents. An effective method is also sought for a wide variety of microorganisms regardless of the environment in which they are bathed.

OBJECT OF THE INVENTION An object of the invention is a method for treating a medium, in particular a liquid medium, comprising microorganisms, the medium being disposed between a first electrode and a second electrode, within an enclosure, - each electrode extending in said enclosure, in contact with said medium, - each electrode being connected to an electrical circuit capable of establishing a bias voltage, at least one frequency, between said electrodes, the method being characterized in that: said frequency varies, in a time interval of less than 1 hour, between a minimum frequency and a maximum frequency, the maximum frequency being at least two times greater than the minimum frequency, so as to inhibit the development of said microorganisms in the middle.

In other words, the bias voltage applied to the electrodes comprises, in a time interval of less than 1 hour, a plurality of frequency components, extending between a minimum frequency and a maximum frequency. The maximum frequency can be at least 2 times, or 10 times, or 100 times or 1000 times greater than the minimum frequency. Said time interval may in particular be less than 30 minutes, or 20 minutes, or 10 minutes, or even 1 minute or 5 seconds or even 1 second. Preferably, said bias voltage simultaneously comprises a plurality of frequency components, between said minimum frequency and said maximum frequency. It can in particular be produced by a white noise generator. Said minimum frequency is preferably greater than 10 Hz. Said bias voltage generates, between the first and the second electrode, an electric field whose maximum amplitude is less than 10 V / cm. Said first and second electrodes are preferably metallic, and comprise a metal, in particular selected from stainless steel, platinum or gold. They can extend one parallel to the other. The medium may in particular comprise an active ingredient capable of inhibiting the growth of microorganisms, preferably obtained from natural plant substances. This medium may especially comprise eugenol, and possibly eugenol acetate. Another object of the invention is a device for carrying out the method as previously described, comprising: an enclosure adapted to receive a medium comprising microorganisms, a first electrode and a second electrode, each electrode being connected to an electrical circuit, the device being characterized in that, the electrical circuit comprises a generator capable of producing, at the terminals of said electrodes, a bias voltage whose frequency varies, in a time interval of less than 1 hour, between a frequency minimum and a maximum frequency, the maximum frequency being at least 2 times greater than the minimum frequency. The electrodes may in particular be made of stainless steel. The generator may comprise a source of current or voltage capable of generating, across said electrodes, a voltage in the form of a white noise.

DESCRIPTION OF THE FIGURES FIG. 1 represents a first example of a device for implementing a method for eliminating bacteria in a liquid medium. Figures 2 and 3 show variants of this example, with electrodes of different geometries.

FIG. 4 represents experimental results showing the evolution of the bacterial concentration as a function of time for various tests carried out. FIG. 5 represents experimental results showing the inhibition of ATP (Adenosine Triphosphate) synthesis as a function of time for various tests carried out.

DETAILED DESCRIPTION FIG. 1 describes a first example of a device capable of implementing the method that is the subject of the invention. An enclosure 11 comprises a medium 10, for example a liquid, and microorganisms 12 bathed in this liquid. The medium may also be an agar. In this example, the microorganisms 12 are Escherichia-type bacteria. Medium 10 is a liquid culture medium called LB (acronym for Luria-Bertani).

Two electrodes 21 and 22 are immersed in the liquid medium 10. In this first example, they have a parallelepipedal shape of length 8 cm, height 3 cm and thickness 0.5 mm. The electrodes are spaced apart by an inter-electrode distance D of 1 cm. The electrodes are, in this example, stainless steel. Other metal electrodes could be usable, for example platinum or gold. Stainless steel appears as a good compromise cost / performance. In addition, stainless steel does not release toxic ions. The electrodes extend parallel to each other. An electrical supply 23 produces an alternating current, so as to generate, between the terminals of the electrodes 21, 22, a potential difference, or alternating polarization voltage U. The medium between the electrodes is then subjected to an alternating electric field É. In order to avoid overheating or the production of toxic ions in the liquid by electrolysis, the frequency of the bias voltage is greater than 10 Hz, and preferably greater than 100 Hz or even 1 kHz. The power supply, or generator, 23 is here a source of white noise, that is to say a source producing a zero average value signal, and whose power spectral density is constant over a given frequency band. The width of the frequency band in which the power spectral density is constant can range from one to three decades or more. Typically, this frequency band extends between 10 kHz and 10 MHz. By constant is meant not deviating from the same value to fluctuations, these fluctuations can reach 10 to 20% of said value. The fact of using such a generator makes it possible to address an extended frequency range, while being of simple design. Therefore, and this is an important element of the invention, the electric field E established between the electrodes 21 and 22 has a multitude of spectral components. The inventors have estimated that the microorganisms present in the liquid medium are likely to be sensitive to at least one of these spectral components, the effect of the electric field E, to this spectral component, being then able to inhibit their development. In the methods of the prior art, a frequency is selected from an extended frequency range, at the end of a learning phase, during which a particular microorganism is successively subjected to different frequencies. exposure to each frequency having a duration of a few hours. This makes it possible to determine the frequency with which the bacteria that one wishes to eliminate is the most sensitive. We can speak of a frequency optimum. With this optimum selected, an electric field at this optimal frequency is then applied to inhibit bacterial growth. The method which is the subject of the invention results from a different approach, since it comprises the application of a signal comprising simultaneously, or in a close time interval, a plurality of spectral components, in an extended spectral band, preferably greater than 1 decade, and typically between 2 and 5 decades, and preferably between 3 and 4 decades. Most of the microorganisms present in the sample are then targeted, without any need to identify them beforehand, and to establish the optimal frequency with respect to each one of them. Moreover, a frequency optimum for a bacterium may depend on the medium in which the latter is placed. By modifying the medium, the frequency optimum can then vary. Since it addresses a wide frequency range, preferably extending over several decades, the method of the invention is more robust with respect to such variations. The white noise generator 23 can easily be manufactured using, for example, a reverse-biased Zener diode in the vicinity of an avalanche voltage.

The signal produced by the Zener diode can then be amplified by a low noise amplifier, or several amplifiers arranged in series, before being applied to the electrodes 21 and 22.

However, even if this is the preferred embodiment, in particular because a white noise generator is simple to produce, it is not necessary that the signal applied to the electrodes is a white noise. Thus, according to a variant, the generator 23 is a generator capable of producing, in a sufficiently short time interval, and preferably simultaneously, an electric current having multiple frequency components, extending over at least one or even two decades, and preferably over at least three decades. In general, the frequency of the signal produced by the generator 23 varies, in a time interval of less than 1 hour, between a minimum frequency (fn ,, n) and a maximum frequency (fniax), the maximum frequency fniax being at least twice or even 10 times, or even at least 100 times greater than the minimum frequency fn ,, n. Considering that the cell division time is between 15 minutes and 30 minutes, a time interval of 1 hour corresponds to a duration greater than twice the cell division time. The method is all the more effective in that the time interval during which the multiple frequencies are applied is small. Thus, the time interval can be reduced to 30 minutes, or 20 minutes, or 10 minutes, or 1 minute, or be less than 5 seconds or even 1 second. FIG. 2 shows another embodiment, in which the electrodes 21 and 22 are placed in a coaxial configuration: the first electrode 21 is rod-shaped, with a diameter of less than 1 cm, for example 0.5 mm, whereas that the second electrode 22 has an annular shape, coaxial with the first electrode. FIG. 3 represents another embodiment, in which the electrodes 21 and 22 are in the form of a "comb" or "interdigitated". Each electrode has a lateral section 21A, 22A of which a plurality of arms 21B, 22B protrude, each arm 25 extending preferably parallel to one another. Preferably, each arm extends perpendicularly to the lateral section to which it is connected. The electrodes 21 and 22 are disposed face to face, so that an arm of an electrode extends between two adjacent arms of the other electrode. This embodiment is suitable for processing larger volumes.

Whatever the embodiment, the distance D between the two electrodes 21 and 22 is preferably centimeter, being between 0.5 cm and 15 or even 20 cm. Beyond this, the bias voltage must increase, knowing that the electric field E between two electrodes preferably has an amplitude of a few V / cm, preferably between 1 and 10 V / cm. Tests Experimental tests were carried out, using a configuration as shown in FIG. 1, the applied bias voltage having a white noise frequency spectrum between 10 kHz and 10 MHz, and an amplitude of 5 V. The 10 electrodes being spaced 1 cm apart, the maximum amplitude of the electric field E between the two electrodes is 5 V / cm. In some configurations, a biocidal product has been added to the culture medium. It is the biocide whose reference is Bioscreen BS 33, supplied by Hightech Bio Activities Holding GMBH. Its composition is described in patent FR2867947. It comprises mainly eugenol, and to a lesser extent eugenol acetate, these components being in particular obtained from clove. The use of such a biocide, obtained with the aid of natural plant substances, limits the harmful effects on the environment. Three configurations are studied: 20 - configuration 1: no treatment: the sample comprises the culture medium as previously described; This is the control pattern; configuration 2: application of the alternating voltage as previously described to the sample; configuration 3: addition of the biocide previously described, diluted with leme, and application of the alternating voltage, analogously to configuration 2. The initial concentration of Escherichia coli bacteria amounts to 5 108 bacteria per ml. The temperature of each sample was maintained at 20 ° C during the tests.

FIG. 4 shows the evolution of the bacterial concentration in the medium, in configurations 2 and 3, as a function of time, the measurement points being made at to, to + 30 min, to + 60 min, to + 120. min, to + 240 min and to + 360 min, to represent the beginning of the experiment, and corresponding to the abscissa 0 in FIG. 4. The abscissas are expressed in minutes while each ordinate represents the bacterial concentration normalized by the concentration in the control sample (configuration 1), at the same time. Each ordinate represents, as a percentage, the evolution of the bacterial population relative to the control sample of the configuration 1. The margins of error are not represented.

The quantification of the bacterial population was carried out by sampling at different times, then dilution and spreading of the sample on agar, before visual counting of the colonies formed 24 to 48 hours after the spreading. Configuration No. 2 (application of an electric field alone) leads to a reduction of the bacterial concentration of about 25% in 6 hours compared to the control sample. Configuration No. 3 (biocide combination and electric field) leads to a reduction in bacterial concentration of about 40% in 6 hours, compared to the control sample. This test shows that the application of an alternating electric field, simultaneously exhibiting spectral components between 10 kHz and 10 MHz, makes it possible to reduce the bacterial growth in a sample after a few hours. Another remarkable point is that the effect of the biocide on bacterial proliferation significantly improves the effect of the electric field. This confirms the synergistic effect between the biocide and the application of an electric field, an effect referred to in the prior art as "bioelectric effect". In addition to enumerating the bacteria by visual method, ATPmetry measurements (luminescence Adenosine triphospate assay) were performed on each sample at to, to + 120 min, to + 240 min and to + 360 min, to representing the beginning of the experiment, and corresponding to the abscissa 0 in Figure 5. After sampling, lysis and pellet recovery, bioluminescence measurements were performed, representative of the concentration of ATP in the sample. These measurements are representative of the energy released by the metabolism of the bacteria contained in the sample.

Figure 5 shows the results of these measurements for the three configurations studied. The x-axis represents the elapsed time since the beginning of the experiment, the y-axis representing, in arbitrary scale, the measured intensity of the luminescence signal. On the control sample (configuration 1), there is an increase in the concentration of ATP, indicating sustained bacterial growth. Configurations 2 and 3 are distinguished by a markedly less marked increase in ATP concentration, reflecting the effect of the electric field alone or, better, in combination with a biocide, for the inhibition of bacterial growth. In addition, the samples were subjected to a temperature control by observation with the aid of a thermal camera. No significant heating was observed. Finally, an ion assay was performed on samples, by ICP-OES (acronym for Inductively Coupled Plasma -Optical Emission Spectrometry, meaning high frequency induced plasma coupling - optical spectrometry). This assay did not reveal a significant increase in the concentration of metal ions in the medium. The method described can be applied to any liquid which it is desired to control or reduce the bacterial proliferation, in the laboratory or on an industrial scale, for example in the agri-food field, the treatment of water, for example domestic water or swimming pool water, or the treatment of other liquid effluents.

On a millimetric or microscopic scale, it may be integrated in microfluidic cards capable of carrying out analyzes or processes on biological samples. The electrodes can be integrated in the board during the manufacture of the board, by using standard microfabrication techniques for the deposition of a metal. In particular, the process can be carried out before biological processes, treatment or analysis, in order to eliminate or reduce the amount of bacteria in the treated sample.

Claims (14)

REVENDICATIONS1. A method of treating a medium (10) comprising microorganisms (12), the medium being disposed between a first electrode (21) and a second electrode (22), within an enclosure (11), - each an electrode extending in said enclosure, in contact with said medium (10), each electrode being connected to an electrical circuit (20) capable of establishing a bias voltage (U), according to at least one frequency (f), between said electrodes , the method being characterized in that: said frequency (f) varies, in a time interval of less than 1 hour, between a minimum frequency (fmn) and a maximum frequency (fmm), the maximum frequency (fmm) being at least 2 times greater than the minimum frequency (fmn), so as to inhibit the development of said microorganisms in the medium. The method of treatment of claim 1, wherein said time interval is less than 30 minutes or 10 minutes or 1 minute or 5 seconds or 1 second. 3. Processing method according to any one of the preceding claims, wherein said bias voltage (U) simultaneously comprises a plurality of frequency components, between said minimum frequency (fmn) and said maximum frequency (fmm). 4. The treatment method as claimed in any one of the preceding claims, wherein said bias voltage (U) is produced by a white noise generator (23). The treatment method according to any one of the preceding claims, wherein the maximum frequency (fmm) is at least ten times or a hundred times or a thousand times greater than the minimum frequency (fmin). 3036708 14 6. Treatment method according to any one of the preceding claims, wherein the minimum frequency (fmn) is greater than 10 Hz. A method of treatment according to any one of the preceding claims, wherein said bias voltage (U) generates, between the first and the second electrode, an electric field (E) whose maximum amplitude is less than 10 V / cm. The treatment method according to any one of the preceding claims, wherein said first and second electrodes are metallic, and comprise a metal selected from stainless steel, platinum or gold. 9. A method of treatment according to any one of the preceding claims, wherein the medium also comprises an active ingredient capable of inhibiting the growth of microorganisms. 10. The treatment method according to claim 9, wherein the active ingredient is obtained from natural plant substances. 11. Apparatus for carrying out the method according to one of the preceding claims, comprising: an enclosure adapted to receive a medium (10) comprising microorganisms, a first electrode (21) and a second electrode (22), each electrode being connected to an electric circuit (20), the device being characterized in that the electrical circuit (20) comprises a generator (23) capable of producing, at the terminals of said electrodes (21, 22), a voltage of polarization whose frequency (f) varies, in a time interval of less than 1 hour, between a minimum frequency (fmn) and a maximum frequency (fmm), the maximum frequency (fmm) being at least 2 times greater than the minimum frequency ( fmn). 25 The device of claim 11, wherein said time interval is less than 20 minutes or 10 minutes or 1 minute. 3036708 15 13. Device according to any one of claims 11 or 12, wherein the generator (23) comprises a current source or voltage capable of generating, across said electrodes, a voltage in the form of a white noise. 14. Device according to any one of claims 11 to 13, characterized in that the electrodes (21,22) are made of stainless steel.